Single domain antibodies directed against intracellular antigens

ABSTRACT

This invention provides compositions and methods to treat a condition or disease without the use of exogenous targeting sequences or chemical compositions. The present invention relates to single-domain antibodies (sdAbs), proteins and polypeptides comprising the sdAbs that are directed against intracellular components that cause a condition or disease. The invention also includes nucleic acids encoding the sdAbs, proteins and polypeptides, and compositions comprising the sdAbs. The invention includes the use of the compositions, sdAbs, and nucleic acids encoding the sdAbs for prophylactic, therapeutic or diagnostic purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 14/922,093 titled “Single Domain Antibodies Directed AgainstSTAT3” filed Oct. 23, 2015, which claims the benefit of U.S. ProvisionalPatent Application No. 62/067,908, filed on Oct. 23, 2014; U.S.Provisional Patent Application No. 62/148,656, filed on Apr. 16, 2015;U.S. Provisional Patent Application No. 62/188,353 filed on Jul. 2,2015; and U.S. Provisional Patent Application No. 62/210,795, filed onAug. 27, 2015, the contents of which are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file titled“Sequence Listing 51293-12” created May 8, 2017, and is 88,000 bytes insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety.

BACKGROUND

The use of single-domain antibodies (sdAbs) as single antigen-bindingproteins or as an antigen-binding domain in larger protein orpolypeptide offers a number of significant advantages over the use ofconventional antibodies or antibody fragments. The advantages of sdAbsinclude: only a single domain is required to bind an antigen with highaffinity and with high selectivity; sdAbs can be expressed from a singlegene and require no post-translational modification; sdAbs are highlystable to heat, pH, proteases and other denaturing agents or conditions;sdAbs are inexpensive to prepare; and sdAbs can access targets andepitopes not accessible to conventional antibodies.

There are a number of diseases or conditions, such as cancer, that arecaused by aberrant intracellular or transmembrane components such asnucleotides and proteins. Elimination of the aberrant components can beused to prevent or treat the diseases or conditions. There are a numberof pharmacological compounds available for treatment, but the compoundscan be ineffective, undeliverable, or toxic to unaffected cells.

Other treatments include the use of therapeutic proteins or agents thatcontain an exogenous targeting sequence so that the therapeutic agentcan be recognized by receptors in the cell membrane, enabling thetherapeutic agent to cross the cell membrane and enter the cell. Oncethe therapeutic agent is inside the cell, the therapeutic agent caninteract with the target component in order to treat the disease.However, the use of exogenous targeting sequence can limit the cell typethat is targeted by the therapeutic agent, and adds to the cost ofmanufacturing the therapeutic agent.

For the foregoing reasons, there is a need for compositions and methodsto treat or prevent a disease that do not rely on exogenous targetingsequences or chemical compositions in order to enter the cell, and thatare effective in targeting only the affected cells in the body.

The present invention relates to single-domain antibodies (sdAbs),proteins and polypeptides comprising the sdAbs. The sdAbs are directedagainst intracellular components that cause a condition or disease. Theinvention also includes nucleic acids encoding the sdAbs, proteins andpolypeptides, and compositions comprising the sdAbs. The inventionincludes the use of the compositions, sdAbs, proteins or polypeptidesfor prophylactic, therapeutic or diagnostic purposes. The invention alsoincludes the use of monoclonal antibodies directed towards the sdAbs ofthe invention.

SUMMARY

The invention includes a single-domain antibody (sdAb) directed againstan intracellular component that can passively cross a cellular membraneto target the intracellular component without exogenous compounds andwithout additional membrane targeting sequences. The intracellularcomponent can be a nucleic acid, protein, lipid, carbohydrate, orcombination thereof. The protein can be either phosphorylated orun-phosphorylated. Additionally, the intracellular component can be oneor more STAT proteins such as STAT1, STAT2, STAT3, STAT4, STAT5a,STAT5b, or STAT6. In one aspect, the sdAb can be multispecific for twoor more antigens.

Another embodiment of the invention is a method of preventing ortreating a disease or disorder, or preventing recurrence of a disease byadministration of one or more of the sdAb of the invention to a subjectin need thereof. In one aspect, the sdAb of the invention can be used todeliver a therapeutic agent. In another aspect, the therapeutic agentcan be delivered across the blood-brain barrier to an individual in needthereof. In yet another aspect, the sdAb of the invention can be used incombination with one or more compounds, such as, for example, atranscriptional inhibitor.

Yet another embodiment of the invention is a sdAb directed against anintracellular component, wherein the sdAb can passively cross theblood-brain barrier to target the intracellular component withoutexogenous compounds and without additional membrane targeting sequences.The intracellular component can be a nucleic acid, protein, lipid,carbohydrate, or combination thereof. The protein can be eitherphosphorylated or un-phosphorylated. Additionally, the intracellularcomponent can be one or more STAT proteins such as STAT1, STAT2, STAT3,STAT4, STAT5a, STAT5b, or STAT6. In one aspect, the sdAb can bemultispecific for two or more antigens.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawings where:

FIG. 1 is a schematic map of VHH13 anti-STAT3 sdAb expression vectorpTT21-stt VHH13;

FIG. 2 is a schematic map of VHH14 anti-STAT3 sdAb expression vectorpTT21-stt VHH14;

FIG. 3 depicts the results of an immunoprecipitation assay usinganti-STAT3 bacterial VHH13 STAT3 (SEQ ID NO:3) and anti-STAT3 bacterialVHH14 STAT3 (SEQ ID NO:4);

FIG. 4 depicts the results of an immunoprecipitation assay usinganti-STAT3 bacterial VHH13 STAT3 (SEQ ID NO:3);

FIG. 5 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the MDA-MB-231 xenograft model, dosed at 0.5mg/kg/day;

FIG. 6 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ. ID. NO. 3) sdAb in MDA-MB-231 xenograft model at doses rangingfrom 1 mg/kg twice daily to 2 mg/kg twice daily or 2 mg/kg/day;

FIG. 7 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the MDA-MB-231 xenograft model, dosed at 5mg/kg/twice daily;

FIG. 8 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the DU145 xenograft model, dosed at 5 mg/kg/twicedaily;

FIG. 9 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the PANC-1 xenograft model, dosed at 5 mg/kg/twicedaily;

FIG. 10 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the MCF-7 xenograft model, dosed at 1 mg/kg/threetimes daily;

FIG. 11 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the BT-474 xenograft model, dosed at 1 mg/kg/threetimes daily;

FIG. 12 illustrates the cytotoxicity of TNF-alpha in U937 cells;

FIG. 13 illustrates the cytotoxicity of Staurosporine in U937 cells;

FIG. 14 illustrates inhibition of TNF-alpha cytotoxicity byanti-TNF-alpha sdAbs;

FIG. 15 shows staining of anti-STAT3 VHH13 (SEQ ID NO:3) sdAb in neuronsand glial cells;

FIG. 16 shows staining of anti-STAT3 VHH13 (SEQ ID NO:3) sdAb in cancercells;

FIG. 17 shows MDA-MB 231 cells incubated with STAT3 VHH13 (SEQ ID NO:3)sdAb;

FIG. 18 shows PANC-1 cells incubated with STAT3 VHH13 (SEQ ID NO:3)sdAb;

FIG. 19 shows MDA-MB 231 cells incubated with TNF-alpha VHH66 (SEQ IDNO:45) sdAb;

FIG. 20 shows PANC-1 cells incubated with TNF-alpha VHH66 (SEQ ID NO:45)sdAb;

FIG. 21 shows cytoplasmic staining of Stat3 in HEp-2 cells incubatedwith STAT3 VHH13 (SEQ ID NO:3) sdAb;

FIG. 22 shows nuclear staining of Stat3 in HEp-2 cells incubated withrecombinant IL-6; and

FIG. 23 shows cytoplasmic staining of Stat3 in HEp-2 cells incubatedwith STAT3 VHH13 (SEQ ID NO:3) sdAb and recombinant IL-6.

DESCRIPTION

As used herein, the following terms and variations thereof have themeanings given below, unless a different meaning is clearly intended bythe context in which such term is used.

The terms “a,” “an,” and “the” and similar referents used herein are tobe construed to cover both the singular and the plural unless theirusage in context indicates otherwise.

The term “antigenic determinant” refers to the epitope on the antigenrecognized by the antigen-binding molecule (such as an sdAb orpolypeptide of the invention) and more in particular by theantigen-binding site of the antigen-binding molecule. The terms“antigenic determinant” and “epitope” may also be used interchangeably.An amino acid sequence that can bind to, that has affinity for and/orthat has specificity for a specific antigenic determinant, epitope,antigen or protein is said to be “against” or “directed against” theantigenic determinant, epitope, antigen or protein.

As used herein, the term “comprise” and variations of the term, such as“comprising” and “comprises,” are not intended to exclude otheradditives, components, integers or steps.

It is contemplated that the sdAbs, polypeptides and proteins describedherein can contain so-called “conservative” amino acid substitutions,which can generally be described as amino acid substitutions in which anamino acid residue is replaced with another amino acid residue ofsimilar chemical structure and which has little or essentially noinfluence on the function, activity or other biological properties ofthe polypeptide. Conservative amino acid substitutions are well known inthe art. Conservative substitutions are substitutions in which one aminoacid within the following groups (a)-(e) is substituted by another aminoacid within the same group: (a) small aliphatic, nonpolar or slightlypolar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negativelycharged residues and their (uncharged) amides: Asp, Asn, Glu and Gln;(c) polar, positively charged residues: His, Arg and Lys; (d) largealiphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e)aromatic residues: Phe, Tyr and Trp. Other conservative substitutionsinclude: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or intoHis; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly intoAla or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leuinto Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu,into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr;Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ileor into Leu.

A “domain” as used herein generally refers to a globular region of anantibody chain, and in particular to a globular region of a heavy chainantibody, or to a polypeptide that essentially consists of such aglobular region.

The amino acid sequence and structure of an sdAb is typically made up offour framework regions or “FRs,” which are referred to as “Frameworkregion 1” or “FR1”; as “Framework region 2” or“FR2”; as “Frameworkregion 3” or “FR3”; and as “Framework region 4” or “FR4,” respectively.The framework regions are interrupted by three complementaritydetermining regions or “CDRs,” which are referred as “ComplementarityDetermining Region 1” or “CDR1”; as “Complementarity Determining Region2” or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3,”respectively.

As used herein, the term “humanized sdAb” means an sdAb that has had oneor more amino acid residues in the amino acid sequence of the naturallyoccurring VHH sequence replaced by one or more of the amino acidresidues that occur at the corresponding position in a VH domain from aconventional 4-chain antibody from a human. This can be performed bymethods that are well known in the art. For example, the FRs of thesdAbs can be replaced by human variable FRs.

As used herein, an “isolated” nucleic acid or amino acid has beenseparated from at least one other component with which it is usuallyassociated, such as its source or medium, another nucleic acid, anotherprotein/polypeptide, another biological component or macromolecule orcontaminant, impurity or minor component.

The term “mammal” is defined as an individual belonging to the classMammalia and includes, without limitation, humans, domestic and farmanimals, and zoo, sports, and pet animals, such as cows, horses, sheep,dogs and cats.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, PBS (phosphate-bufferedsaline), and 5% human serum albumin. Liposomes, cationic lipids andnon-aqueous vehicles such as fixed oils may also be used. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with a therapeutic agent as defined above, use thereof inthe composition of the present invention is contemplated.

A “quantitative immunoassay” refers to any means of measuring an amountof antigen present in a sample by using an antibody. Methods forperforming quantitative immunoassays include, but are not limited to,enzyme-linked immunosorbent assay (ELISA), specific analyte labeling andrecapture assay (SALRA), liquid chromatography, mass spectrometry,fluorescence-activated cell sorting, and the like.

The term “solution” refers to a composition comprising a solvent and asolute, and includes true solutions and suspensions. Examples ofsolutions include a solid, liquid or gas dissolved in a liquid andparticulates or micelles suspended in a liquid.

The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular antigen-bindingmolecule or antigen-binding protein molecule can bind. The specificityof an antigen-binding protein can be determined based on affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (KD), is ameasure for the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the KD, the stronger the binding strength between an antigenicdeterminant and the antigen-binding molecule (alternatively, theaffinity can also be expressed as the affinity constant (KA), which is1/KD). As will be clear to one of skill in the art, affinity can bedetermined depending on the specific antigen of interest. Avidity is themeasure of the strength of binding between an antigen-binding moleculeand the antigen. Avidity is related to both the affinity between anantigenic determinant and its antigen binding site on theantigen-binding molecule and the number of pertinent binding sitespresent on the antigen-binding molecule. Specific binding of anantigen-binding protein to an antigen or antigenic determinant can bedetermined by any known manner, such as, for example, Scatchard analysisand/or competitive binding assays, such as radioimmunoassays (RIA),enzyme immunoassays (EIA) and sandwich competition assays.

As used herein, the term “recombinant” refers to the use of geneticengineering methods (for example, cloning, and amplification) used toproduce the sdAbs of the invention.

A “single domain antibody,” “sdAb” or “VHH” can be generally defined asa polypeptide or protein comprising an amino acid sequence that iscomprised of four framework regions interrupted by three complementaritydetermining regions. This is represented asFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An sdAb of the invention also includes apolypeptide or protein that comprises the sdAb amino acid sequence.Typically, sdAbs are produced in camelids such as llamas, but can alsobe synthetically generated using techniques that are well known in theart. As used herein, the variable domains present in naturally occurringheavy chain antibodies will also be referred to as “VHH domains,” inorder to distinguish them from the heavy chain variable domains that arepresent in conventional 4-chain antibodies, referred to as “VH domains,”and from the light chain variable domains that are present inconventional 4-chain antibodies, referred to as “VL domains.” “VHH” and“sdAb” are used interchangeably herein. The numbering of the amino acidresidues of a sdAb or polypeptide is according to the general numberingfor VH domains given by Kabat et al. (“Sequence of proteins ofimmunological interest,” US Public Health Services, NIH Bethesda, Md.,Publication No. 91). According to this numbering, FR1 of a sdAbcomprises the amino acid residues at positions 1-30, CDR1 of a sdAbcomprises the amino acid residues at positions 31-36, FR2 of a sdAbcomprises the amino acids at positions 36-49, CDR2 of a sdAb comprisesthe amino acid residues at positions 50-65, FR3 of a sdAb comprises theamino acid residues at positions 66-94, CDR3 of a sdAb comprises theamino acid residues at positions 95-102, and FR4 of a sdAb comprises theamino acid residues at positions 103-113.

The term “synthetic” refers to production by in vitro chemical orenzymatic synthesis.

The term “target” as used herein refers to any component, antigen, ormoiety that is recognized by the sdAb. The term “intracellular target”refers to any component, antigen, or moiety present inside a cell. A“transmembrane target” is a component, antigen, or moiety that islocated within the cell membrane. An “extracellular target” refers to acomponent, antigen, or moiety that is located outside of the cell.

A “therapeutic composition” as used herein means a substance that isintended to have a therapeutic effect such as pharmaceuticalcompositions, genetic materials, biologics, and other substances.Genetic materials include substances intended to have a direct orindirect genetic therapeutic effect such as genetic vectors, geneticregulator elements, genetic structural elements, DNA, RNA and the like.Biologics include substances that are living matter or derived fromliving matter intended to have a therapeutic effect.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management of adisease or an overt symptom of the disease. The therapeuticallyeffective amount may treat a disease or condition, a symptom of disease,or a predisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, or affect thedisease, the symptoms of disease, or the predisposition toward disease.The specific amount that is therapeutically effective can be readilydetermined by an ordinary medical practitioner, and may vary dependingon factors known in the art, such as, e.g., the type of disease, thepatient's history and age, the stage of disease, and the administrationof other therapeutic agents.

The present invention relates to single-domain antibodies (sdAbs) thatare directed against intracellular components, as well as to proteinsand polypeptides comprising the sdAbs and nucleotides encoding theproteins and polypeptides. The invention can also relate to sdAbs thatare directed against intercellular, transcellular and extracellulartargets or antigens. The invention also includes nucleic acids encodingthe sdAbs, proteins and polypeptides, and compositions comprising thesdAbs. The invention includes the use of the compositions, sdAbs,proteins or polypeptides for prophylactic, therapeutic or diagnosticpurposes.

SdAbs have a number of unique structural characteristics and functionalproperties which make sdAbs highly advantageous for use as functionalantigen-binding domains or proteins. SdAbs functionally bind to anantigen in the absence of a light chain variable domain, and canfunction as a single, relatively small, functional antigen-bindingstructural unit, domain or protein. This distinguishes sdAbs from thedomains of conventional antibodies, which by themselves do not functionas an antigen-binding protein or domain, but need to be combined withconventional antibody fragments such as Fab fragments or ScFv's fragmentin order to bind an antigen.

SdAbs can be obtained using methods that are well known in the art. Forexample, one method for obtaining sdAbs includes (a) immunizing aCamelid with one or more antigens, (b) isolating peripheral lymphocytesfrom the immunized Camelid, obtaining the total RNA and synthesizing thecorresponding cDNAs, (c) constructing a library of cDNA fragmentsencoding VHH domains, (d) transcribing the VHH domain-encoding cDNAsobtained in step (c) to mRNA using PCR, converting the mRNA to ribosomedisplay format, and selecting the VHH domain by ribosome display, and(e) expressing the VHH domain in a suitable vector and, optionallypurifying the expressed VHH domain.

Another method of obtaining the sdAbs of the invention is by preparing anucleic acid encoding an sdAb using techniques for nucleic acidsynthesis, followed by expression of the nucleic acid in vivo or invitro. Additionally, the sdAb, polypeptides and proteins of theinvention can be prepared using synthetic or semi-synthetic techniquesfor preparing proteins, polypeptides or other amino acid sequences.

The sdAbs of the invention will generally bind to all naturallyoccurring or synthetic analogs, variants, mutants, alleles, parts andfragments of the target, or at least to those analogs, variants,mutants, alleles, parts and fragments of the target that contain one ormore antigenic determinants or epitopes that are essentially the same asthe antigenic determinant or epitope to which the sdAbs of the inventionbind in the wild-type target. The sdAbs of the invention may bind tosuch analogs, variants, mutants, alleles, parts and fragments with anaffinity and/or specificity that is the same as, or that is higher thanor lower than the affinity and specificity with which the sdAbs of theinvention bind to the wild-type target. It is also contemplated withinthe scope of the invention that the sdAbs of the invention bind to someanalogs, variants, mutants, alleles, parts and fragments of the targetbut not to others. In addition, the sdAb of the invention may behumanized, and may be monovalent or multivalent, and/or multispecific.Additionally, the sdAbs of the invention can bind to the phosphorylatedform of the target protein as well as the unphosphorylated form of thetarget protein. SdAbs can be linked to other molecules such as albuminor other macromolecules.

In addition, it is within the scope of the invention that the sdAbs aremultivalent, that is, the sdAb can have two or more proteins orpolypeptides which are directed against two or more different ofepitopes of the target. In such a multivalent sdAb, the protein orpolypeptide may be directed, for example, against the same epitopes,substantially equivalent epitopes, or different epitopes. The differentepitopes may be located on the same target, or it could be on two ormore different targets.

It is also contemplated that the sequence of one or more sdAbs of theinvention may be connected or joined with one or more linker sequences.The linker can be, for example, a protein sequence containing acombination of serines, glycines and alanines.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the sdAbs ofthe invention, as long as these are suitable for the described uses.

Since the sdAbs of the invention are mainly intended for therapeuticand/or diagnostic use, they are directed against mammalian, preferablyhuman, targets. However, it is possible that the sdAbs described hereinare cross-reactive with targets from other species, for example withtargets from one or more other species of primates or other animals (forexample, mouse, rat, rabbit, pig or dog), and in particular in animalmodels for diseases and disorders associated with the disease associatedwith the targets.

In another aspect, the invention relates to a nucleic acid that encodesan sdAb of the invention. Such a nucleic acid may be, for example, inthe form of a genetic construct.

In another aspect, the invention relates to host or host cell thatexpresses or is capable of expressing an sdAb of the invention, and/orthat contains a nucleic acid encoding a sdAb of the invention. Sequencesof the sdAbs can be used to insert into the genome of any organism tocreate a genetically modified organism (GMO). Examples include, but arenot limited to, plants, bacteria, viruses, and animals.

The invention further relates to methods for preparing or generating thesdAbs, nucleic acids encoding the sdAbs, host cells expressing orcapable of expressing such sdAbs, products and compositions containingthe sdAbs of the invention.

The invention further relates to applications and uses of the sdAb, thenucleic acids encoding the sdAbs, host cells, products and compositionsdescribed herein. Such a product or composition may, for example, be apharmaceutical composition for treatment or prevention of a disease, ora product or composition for diagnostic use. The sdAb of the inventioncan also be used to deliver a therapeutic agent or can be covalentlylinked to a molecule in order to deliver the therapeutic agent into acell or across the blood-brain barrier of a person in need thereof. ThesdAb can be used to target primary malignancies of the central nervoussystem, metastatic cancer, and central nervous system diseases such asmultiple sclerosis, dementia, and the like. Furthermore, sdAbs can beused in a variety of assays, for example ELISA assays and massspectrometry assays to measure the serum and tissue levels of the sdAbs.

In another aspect, a nucleic acid encoding one or more sdAb of theinvention can be inserted into the genome of an organism to treat orprevent diseases.

The present invention generally relates to sdAbs, as well as to proteinsor polypeptides comprising or essentially consisting of one or more ofsuch sdAbs, that can be used for prophylactic, therapeutic and/ordiagnostic purposes.

The methods and compositions detailed in the present invention can beused to treat disease described herein, and can be used with any dosageand/or formulation described herein or otherwise known, as well as withany route of administration described herein or otherwise known to oneof skill in the art.

The sdAbs of the invention, in particular the anti-STAT3 VHH, theanti-KRAS VHH, and the anti-TNF-alpha VHH of the present invention, canbe used for treatment and prevention of malignant diseases including,but not limited to: multiple myeloma, leukemias (HTLV-1 dependent,erythroleukemia, acute myelogenous leukemia (AML), chronic myelogenousleukemia (CML), and large granular lymphocyte leukemia (LGL), lymphomas(EBV-related/Burkitt's, mycosis fungoides, cutaneous T-cell lymphoma,non-Hodgkins lymphoma (NHL), anaplastic large-cell lymphoma (ALCL),breast cancers, triple-negative breast cancers, head and neck cancers,melanoma, ovarian cancers, lung cancers, pancreatic cancers, prostatecancers, sarcomas, osteosarcoma, Kaposi's sarcoma, Ewing's sarcoma,hepatocellular cancers, glioma, neuroblastoma, astrocytoma, colorectalcancers, Wilm's tumors, renal cancers, bladder cancers, endometrialcancers, cervical cancers, esophageal cancers, cutaneous squamous cellcancers, basal cell cancers, and any metastatic cancers. The sdAbs canbe used in cancer patients to help prevent or reduce weight loss orcachexia due to cancer.

The sdAb, in particular the anti-STAT3 and the anti-TNF-alpha sdAbs ofthe present invention, can also be used for treatment and prevention ofdiseases such as, but not limited to: autoimmune diseases (e.g.,rheumatoid arthritis, ulcerative colitis, Crohn's disease, bacterialinduced colitis, asthma, scleroderma, lupus, encephalomyelitis,arteritis, vasculitis, glomerulonephritis, uveitis, uveoretinitis,multiple sclerosis), polycystic kidney disease, dermatologic diseases(e.g., psoriasis, alopecia areata, atopic dermatitis,keloids/hypertrophic scars, lipoma, Paget's disease, and actinickeratosis), Hidradenitis suppurativa, transplantation (e.g., solidorgan, bone marrow, hand, face, limbs, any body part), musculardystrophy and muscle wasting associated with cancers and aging,endometriosis, macular degeneration, retinal degeneration, stroke,epilepsy, traumatic brain and spinal cord injuries, hypertension,cardiac hypertrophy, Alzheimer's disease, pulmonary artery hypertension,type 2 diabetes mellitus, and ankylosing spondylitis. Additionally,sdAbs can target orphan diseases. Examples of these rare orphan diseasesinclude, but are not limited to, triple negative breast cancers,pancreatic cancers, AML (acute myeloid leukemia), head and neck cancers,multiple myeloma, and chemo-resistant cancers.

Viral infections can be treated by targeting intracellular viralproteins in infected cells. Viral proteins, such as HIV reversetranscriptase, can block viral life-cycle. The sdAb of the invention canalso target intracellular viral proteins such as Ebola VP24 and thusblock Ebola's ability to shut down the host's anti-viral immuneresponse. The sdAbs of the invention can be used to target diseases whenthere is an overexpression of an intracellular molecule. Huntington'sdisease can be treated with sdAbs.

The sdAbs of the invention can be used with one or more compounds. Forexample, the sdAb of the invention can be used with JAK/STAT inhibitorssuch as, for example, Curcumin, Resveratrol, Cucurbitacin A, B, E, I, Q,Flavopiridol, Deoxytetrangomycin, Cyclopentenone derivatives,N-Acylhomoserine Lactone, Indirubin derivatives, Meisoindigo,Tyrphostins, Platinum-containing compounds (e.g., IS3-295),Peptidomimetics, antisense oligonucleotides, S3I-201, phosphotyrosintripeptide derivatives, HIV protease inhibitors (e.g., nelfinavir,indinavir, saquinavir, & ritornavir), JSI-124, XpYL, Ac-pYLPQTV-NH2, ISS610, CJ-1383, pyrimethamine, Metformin, Atiprimod, S3I-M2001, STX-0119;N-[2-(1,3,4-oxadiazolyl)]-4 quinolinecarboxamide derivative, S3I-1757,LYS;5,8-dioxo-6(pyridin-3-ylamino)-5,8,-dihydro-naphthalene-1-sulfonamide,withacinstin, Stattic, STA-21, LLL-3, LLL12, XZH-5, SF-1066, SF-1087,17o, Cryptotanshinone, FLL32, FLL62, C188-9, BP-1108 and BP-1075,Galiellalactone, JQ1, 5, 15 DPP, WP1066, Niclosamide, SD1008,Nifuroxazide, Cryptotanshinone, BBI quinone, and Ruxolitnib Phosphate.The one or more compounds can increase the therapeutic response andaugment the effectiveness of the sdAb of the invention. In addition, theeffectiveness of the sdAb can be increased by combining it withpeptides, peptidomimetics, and other drugs, such as, for example, butnot limited to, cimetidine, atorvastatin, celecoxib, metformin, andcimetidine. In addition, anti-STAT3 sdAbs can convert radioresistantcancers to radiosensitive cancers with respect to radiation therapy.

It is also contemplated that one or more sdAbs of the invention can becombined, or the sdAbs of the invention can be combined with othersdAbs.

It is contemplated that certain sdAbs of the invention can cross thecell membrane and enter the cell without the aid of additional targetingprotein sequences on the sdAb, and without the aid of exogenouscompounds that direct the sdAb to bind to the cell surface receptors andcross the cell membrane. Additionally, sdAbs of the invention can crossthe blood-brain barrier.

After crossing the cell membrane, these sdAbs can target transmembraneor intracellular molecules or antigens. These intracellular ortransmembrane targets can be, for example, proteins, carbohydrates,lipids, nucleic acids, mutated proteins, viral proteins, and prions. ThesdAb targets may function as enzymes, structural proteins of the cell,intracellular portions of cell membrane molecules, molecules within themembranes of organelles, any type of RNA molecule, any regions of DNA orchromosome, methylated or unmethylated nucleic acids, partiallyassembled molecules within the synthesis mechanism of the cell, secondmessenger molecules, and molecules within cell signaling mechanisms.Targets may include all molecules in the cytoplasm, nucleus, organelles,and cell membrane. Molecules destined for secretion or placement in thecell membrane can be targeted within the cytoplasm before leaving thecell.

The sdAbs of the invention can cross the blood-brain barrier and targetbrain cells in vivo without exogenous compounds. The sdAbs of theinvention can also be used as carriers to transport therapeutics orother molecules across the blood-brain barrier.

The sdAb targets can be in humans, animals, plants, fungi, parasites,protists, bacteria, viruses, prions, prokaryotic cells, and eukaryoticcells. Some examples of inter- and intracellular signaling molecules andprotein groups that can be targeted by the sdAbs of the invention are:oncogene products, hormones, cytokines, growth factors,neurotransmitters, kinases (including tyrosine kinase, serine kinase,and threonine kinase), phosphatases, ubiquitin, cyclic nucleotides,cyclases (adenylyl and guanylyl), G proteins, phosphodiesterases, GTPasesuperfamily, immunoglobulins (antibodies, Fab fragments, binders,sdAbs), immunoglobulin superfamily, inositol phosphate lipids, steroidreceptors, calmodulin, CD group (e.g., CD4, CD8, CD28, etc.),transcription factors, TGF-beta, TNF-alpha and beta, TNF ligandsuperfamily, notch receptor signaling molecules, hedgehog receptorsignaling molecules, Wnt receptor signaling molecules, toll-likereceptor signaling molecules, caspases, actin, myosin, myostatin,12-lipoxygenase, 15-lipoxygenase, lipoxygenase superfamily, reversetranscriptase, viruses and their proteins, amyloid proteins, collagen, Gprotein coupled receptors, mutated normal proteins, prions, Ras, Raf,Myc, Src, BCR/ABL, MEK, Erk, Mos, Tp12, MLK3, TAK, DLK, MKK, p38, MAPK,MEKK, ASK, SAPK, JNK, BMK, MAP, JAK, PI3K, cyclooxygenase, STAT1, STAT2,STAT3, STAT4, STAT5a, STAT5b, STAT6, Myc, p53, BRAF, NRAS, KRAS, HRASand chemokines. IL-6 production can also be regulated by the sdAbs ofthe invention. Increased IL-6 production can result in cell growth andwound healing, while decreased IL-6 production can result in decreasedcell proliferation.

KRAS is a Kirsten ras oncogene homolog from the mammalian ras genefamily. KRAS encodes a protein that is a member of the small GTPasesuperfamily. The protein is implicated in various malignancies,including lung adenocarcinoma, mucinous adenoma, ductal carcinoma of thepancreas, and colorectal carcinoma. Under normal conditions, Ras familymembers influence cell growth and differentiation events in asubcellular membrane compartmentalization-based signaling system.However, oncogenic Ras can deregulate processes that control both cellproliferation and apoptosis.

Anti-KRAS sdAbs were developed to target wild-type and mutated KRAS(G12D) in order to disrupt its role in malignant cells such as, forexample, cells involved in colorectal cancer, pancreatic cancer, biliarytract cancer, lung cancer, leukemias, and other metastatic malignancies.Without being bound by a particular mechanism, it is thought that theanti-KRAS sdAb binds KRAS and blocks the downstream signaling of KRAS inmalignant cells. Additionally, the anti-KRAS sdAb may successfully treatmalignancies that are resistant to anti-EGFR biologics (e.g., cetuximaband panitumumab).

Using methods that are well-known in the art, recombinant human mutantKRAS (G12D) protein was used to generate sdAbs that are directed againstor can bind to an epitope of KRAS or mutant KRAS (G12D), or other KRASmutants. Additionally, sdAbs can be generated to other KRAS mutants. Togenerate the anti-KRAS sdAbs, recombinant full-length human KRAS (GeneID: 3845) was expressed in Escherichia coli.

Several sdAbs were obtained and screened. The DNA sequence of oneanti-KRAS (G12D) sdAb, named KRAS_13 (SEQ ID NO:1), is shown below:

5′Gaggtgcagctggtggagtctgggggaggctcggtgcagactggagggtctctgagactctcctgtgcagtttctggaaatatcggcagcagctactgcatgggctggttccgccaggctccagggaagaagcgcgaggcggtcgcacgtattgtacgtgatggtgccactggctacgcagactacgtgaagggccgattcaccatctcccgagacagcgccaagaacactctgtatctgcaaatgaacaggctgatacctgaggacactgccatctactactgtgcggcagacctgcccccaggttgtttgactcaggcgatttggaattttggttatcggggccagggaaccctggtcaccgtctcctca-3′

The amino acid sequence of the anti-KRAS (G12D) sdAb (SEQ ID NO:2),KRAS_13, is shown below, with the CDRs underlined:

EVQLVESGGGSVQTGGSLRLSCAVSGNIGSSYCMGWFRQAPGKKREAVARIVRDGATGYADYVKGRFTISRDSAKNTLYLQMNRLIPEDTAIYYCAADLPPGCLTQAIWNFGYRGQGTLVTVSS

Additionally, the present invention comprises one or more mousemonoclonal antibodies which are directed against one or more domains ofthe anti-KRAS sdAb of the invention. The mouse monoclonal antibody canbe generated by methods that are known by one of skill in the art, forexample, the mouse monoclonal antibody can be produced by a mousehybridoma. The mouse monoclonal antibody can be used in diagnosticassays, for example, the antibody can be used in an immunoassay such asan ELISA or mass spectrometry assay in order to measure the amount ofanti-KRAS sdAb present in a patient's serum. The cytotoxicity of KRAS(G12D) sdAbs on PANC-1 human pancreatic cancer cells was tested, asdescribed below.

STAT3 is a member of the signal transducers and activators oftranscription (STAT) family of proteins that carry both signaltransduction and activation of transcription functions. STAT3 is widelyexpressed and becomes activated through phosphorylation on tyrosineand/or serine as a DNA binding protein in response to a variouscytokines and growth factors such as EGF, IL-6, PDGF, IL-2 and G-CSF.The STAT3 phosphoprotein forms homodimers and heterodimers with othermembers of the STAT family and translocates to the nucleus in order tomodulate the transcription of various genes, and as a result plays a keyrole in many cellular processes such as cell growth, apoptosis,angiogenesis, immune evasion, and survival.

An anti-STAT3 sdAb can be given to patients and other organisms to treatdiseases caused by phosphorylated and non-phosphorylated STAT3, as wellas to prevent the development of disease or recurrence of disease. Forexample, patients who have undergone organ transplant and bone marrowtransplant are at higher risk for cutaneous SCCA and BCCA due to theimmunosuppressive medications they take. Administration of an anti-STAT3sdAb can reduce or eliminate this risk. Patients treated for amalignancy who are at risk for recurrence will benefit from treatmentwith the anti-STAT3 sdAb. Based on family medical history and HLA-type,some individuals will be at increased risk for some types of autoimmunediseases and may benefit from treatment with sdAbs to reduce risk ofdeveloping that autoimmune disease. Breast cancer risk can be reducedwith administration of anti-STAT3 medication such as GLG-302, asdemonstrated in a recent NCI study.

In addition to inhibiting STAT3, the anti-STAT3 sdAb can also inhibitSTAT1, STAT2, STAT4, STAT5a, STAT5b, and STAT6 due to the high degree ofhomology between these molecules.

Recombinant human STAT3 protein was used to produce anti-STAT sdAbs thatwere directed against or can bind to an epitope of STAT3. To generatethe anti-STAT3 sdAbs, recombinant full-length human STAT3 (Gene ID:6774) was expressed by baculovirus in Sf9 insect cells. The anti-STATsdAbs were cloned into vectors that can be expressed in both bacterialand mammalian cells, as shown in FIGS. 1 and 2.

The anti-STAT3 sdAb of the invention can be used to target STAT3 and allother STAT molecules inside the cell in order to inhibit cell growth,such as, for example, suppression of cancer cell growth. In addition,the anti-STAT3 sdAb can inhibit cell growth in other proliferativediseases such as psoriasis, diabetic retinopathy, and maculardegeneration via inhibition of the production of VEGF.

Without being limited to a particular mechanism of action, it is thoughtthat anti-STAT3 sdAb can eliminate cancer induced immune suppression bydecreasing STAT3 levels in antigen presenting cells such as, forexample, host dendritic cells. STAT3 inhibition promotes anti-cancerresponse by patient's innate and adaptive immune systems (i.e.,dendritic cells, macrophages, neutrophils, T cells, NK cells, and Bcells).

Using methods that are well known in the art, several anti-STAT sdAbswere obtained and screened for the ability to suppress cancer cellgrowth and induce apoptosis in cancer cell lines, as described below.The cytotoxicity and anti-proliferative activities of the anti-STAT3sdAbs was tested. In addition, the tolerance of anti-STAT3 sdAbs wastested in vitro and in vivo. The production of mouse monoclononalantibody directed against one or more domains of the anti-STAT sdAbs isdescribed below.

The amino acid sequence of one anti-STAT3 sdAb, named VHH13 (SEQ IDNO:3), is shown below:

HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSGISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSRFDCYRGSWFNRYMYNSWGQGTQVTVSSThe three CDRs are underlined.

The amino acid sequence of a second anti-STAT3 sdAb, named VHH14 (SEQ IDNO:4), is shown below:

QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAALSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAAREGWECGETWLDRTAGGHTYWGQGTLVTVSS

Again, the three CDRs are underlined. The protein sequences of otheranti-STAT3 sdAbs that were obtained are as follows:

STAT3_10 (SEQ ID NO: 5): (1) DVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAA (48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDA AMYYCAARE (98)GWECGETWLDRTAGGHTYWGQGTQVTVSS STAT3_34 (SEQ ID NO: 6): (1)DVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKE REGVAA (48)LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDA AMYYCAARE (98)GWECGETWLDRTAGGHTYWGQGTQVTVSS STAT3_19 (SEQ ID NO: 7): (1)HVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKE REGVAA (48)LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDA AMYYCAARE (98)GWECGETWLDRTAGGHTYWGQGTQVTVSS STAT3_14 (SEQ ID NO: 8): (1)QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKE REGVAA (48)LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDA AMYYCAARE (98)GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_35 (SEQ ID NO: 9): (1)QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKE REGVAA (48)LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDA AMYYCAARE (98)GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_9 (SEQ ID NO: 10): (1)QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKE REGVAA (48)LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDA AMYYCAARE (98)GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_30 (SEQ ID NO: 11): (1)QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKE REGVAA (48)LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDA AMYYCAARE (98)GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_23 (SEQ ID NO: 12): (1)QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKE REGVAA (48)LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDA AMYYCAARE (98)GWECGETWLDRTAGSHTYWGQGTLVTVSS STAT3_24 (SEQ ID NO: 13): (1)EVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKE REGVAA (48)LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDA AMYYCAARE (98)GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_36 (SEQ ID NO: 14): (1)DVQLVESGGGSVQAGDSLRLSCVASTYTGCMGWFRQAPGKE REGVAA (48)LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDA AMYYCAARE (98)GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_12 (SEQ ID NO: 15): (1)QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_16 (SEQ ID NO: 16): (1)QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTNNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_11 (SEQ ID NO: 17): (1)EVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_20 (SEQ ID NO: 18): (1)DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_2 (SEQ ID NO: 19): (1)DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_15 (SEQ ID NO: 20): (1)DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_6 (SEQ ID NO: 21): (1)HVQLVESEGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_33 (SEQ ID NO: 22): (1)QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_17 (SEQ ID NO: 23): (1)QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_25 (SEQ ID NO: 24): (1)EVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMSSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_32 (SEQ ID NO: 25): (1)DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_13 (SEQ ID NO: 26): (1)HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_39 (SEQ ID NO: 27): (1)HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_4 (SEQ ID NO: 28): (1)HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_29 (SEQ ID NO: 29): (1)HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVP GKEREGVSG (51)ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDT AMYYCATSR (101)FDCYRGSWFNRYMYNSWGQGTQVTVSS

The corresponding anti-STAT3 DNA sequences are as follows:

Stat3_VHH-10 (SEQ ID NO: 30):5′-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggacccaggtcaccgtctcctca-3′ Stat3_VHH-14 (SEQ ID NO: 31):5′-caggtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-12 (SEQ ID NO: 32):5′-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-13 (SEQ ID NO: 33):5′-catgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaacggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcactgtctcctca-3′ Stat3_VHH-20 (SEQ ID NO: 34):5′-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-23 (SEQ ID NO: 35):5′-caggtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagcagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggagccatacctactggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-24 (SEQ ID NO: 36):5′-gaggtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccgaaccgccgggggccatacctactggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-25 (SEQ ID NO: 37):5′-gaggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggtcgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgagcagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcaccgtctcctca-3′ Stat3_VHH-19 (SEQ ID NO: 38):5′-catgtgcagctggtggagtctggggggggctcggtgcaggctggaggctctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggacccaggtcaccgtctcctca-3′ Stat3_VHH-32 (SEQ ID NO: 39):5′-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcaccgtctcctca-3′ Stat3_VHH-33 (SEQ ID NO: 40):5′-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcaccgtctcctca-3′ Stat3_VHH-36 (SEQ ID NO: 41):5′-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagactctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggaccctggtcactgtctcctca-3′ Stat3_VHH-11 (SEQ ID NO: 42):5′-gtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgtgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcactgtctcctca-3′ Stat3_VHH-6 (SEQ ID NO: 43):5′-gtgcagctggtggagtctgagggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-1 (SEQ ID NO: 44):5′-gtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaataacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcactgtctcctca-3′

Additionally, the present invention comprises one or more mousemonoclonal antibodies which are directed against one or more domains ofthe anti-STAT3 sdAb of the invention. The mouse monoclonal antibody canbe generated by methods that are known by one of skill in the art, forexample, the mouse monoclonal antibody can be produced by a mousehybridoma. The mouse monoclonal antibody can be used in diagnosticassays, for example, the antibody can be used in an immunoassay such asan ELISA in order to measure the amount of anti-STAT3 sdAb present in apatient's serum. It should be appreciated that the method is not limitedto anti-STAT3 sdAbs, and could be used to produce a mouse antibodydirected towards any of the sdAbs of the present invention.

Anti-STAT5a sdAbs were also generated. The DNA and corresponding proteinsequences for one clone is listed below.

Stat5-31 (SEQ ID NO: 82)5′-gaggtgcagctggtggagtctgggggaggctcggtgcagactggagggtctctgagactctcctgcgcagcctctggattcccctttagtagtcacgttatgggctggttccgccaggctccagggaagaaacgcgagggggtcgcagctatttcggttgatagtggtagcacatggtatgccgactccgtgaagggccgattcaccatctccctggacagcgccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgactagacgtggagttattcttacactaagcccagagacctatgactactggggccaggggacccaggtcaccgtctcctca-3′ STAT5-31 (SEQ ID NO: 83)EVQLVESGGGSVQTGGSLRLSCAASGFPFSSHVMGWFRQAPGKKREGVAAISVDSGSTWYADSVKGRFTISLDSAKNTLYLQMNSLKPEDTAMYYCATRRGVILTLSPETYDYWGQGTQVTVSS

The TNF-alpha gene encodes a multifunctional proinflammatory cytokinethat belongs to the tumor necrosis factor (TNF) superfamily. Thiscytokine is mainly secreted by macrophages. The cytokine is involved inthe regulation of a wide spectrum of biological processes includinggrowth regulation, differentiation, inflammation, viral replication,tumorigenesis, and autoimmune diseases; and in viral, bacterial, fungal,and parasitic infections. Besides inducing hemorrhagic necrosis oftumors, TNF was found to be involved in tumorigenesis, tumor metastasis,viral replication, septic shock, fever, inflammation, cachexia, andautoimmune diseases including Crohn's disease, and rheumatoid arthritisas well as graft-versus-host disease.

The present invention provides sdAbs, proteins, and polypeptides thatare directed against TNF-alpha, in particular against human TNF-alphainside the cell or cell membrane, so as to prevent the secretion ofTNF-alpha by cells.

It is contemplated that the anti-TNF-alpha sdAbs and polypeptides of theinvention can be used for the prevention and/or treatment of diseasesand disorders associated with and/or mediated by TNF-alpha, such asinflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis,inflammatory bowel syndrome, multiple sclerosis, Addison's disease,autoimmune hepatitis, autoimmune parotitis, diabetes type 1,epididymitis, glomerulonephritis, Graves' disease, Guillain-Barresyndrome, Hashimoto's disease, hemolytic anemia, systemic lupuserythematosus, male infertility, multiple sclerosis, myasthenia gravis,pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies,thyroiditis, vasculitis, and weight loss due to cancer and cachexia.

TNF-alpha exists in different forms; there are monomeric and multimericforms, including a trimeric form. It is within the scope of theinvention that the sdAbs, proteins and polypeptides of the inventionbind to TNF-alpha in its different form, i.e., monomeric form ormultimeric forms. Thus, when sdAbs, proteins and polypeptides of theinvention are directed to TNF-alpha, it should be understood that thisalso comprises sdAbs, proteins and polypeptides directed againstTNF-alpha in its trimeric form.

It is known that signal transduction by TNF involves crosslinking by TNFreceptors by a trimer of TNF molecules, which contains three receptorbinding sites (see, for example, Peppel et al., J. Exp. Med., 174(1991), 1483-1489).

Recombinant human TNF-alpha protein was used to generate sdAbs that aredirected against or can bind to an epitope of TNF-alpha. To generate theanti-TNF-alpha sdAbs, recombinant full-length human TNF-alpha (Gene ID:7124) was expressed in Escherichia coli and used as the target antigen.

Thirty-five sdAbs against the TNF-alpha protein were obtained. Theseanti-TNF-alpha antibodies were divided into three groups based onsequence homology.

The amino acid sequence of the first anti-TNF-alpha sdAb, namedTNF-alpha VHH66 (SEQ ID NO:45) sdAb, is shown below:

HVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGLTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRG-QGTLVTVSSThe three CDRs are underlined.

The amino acid sequence of the second anti-TNF-alpha sdAb, namedTNF-alpha VHH69 (SEQ ID NO:46) sdAb, is shown below:

EVQLVESGGGSVLAGGSLRLSCVASGFTSRYNYMAWFRQAPGKEREGVATIGTASGSADYYGSVKDRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAARTYGTISLTPSDYRYWGQGTLVTVSSThe three CDRs are underlined.

The amino acid sequence of the third anti-TNF-alpha sdAb, namedTNF-alpha VHH62 (SEO ID NO:471 sdAb, is shown below:

QVQLVESGGGPVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCSWAQGTQGTLVTVSS

The three CDRs are underlined. Other anti-TNF-alpha sdAbs that werefound include the sequences below, again with the CDRs underlined:

TNF_2 (SEQ ID NO: 48): QVQLVESGGGSVEAGRSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_46 (SEQ ID NO: 49):QVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGQTTHADSVKGRFTISRDNVKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_71 (SEQ ID NO: 50):QVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGLTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_21 (SEQ ID NO: 51):QVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_38 (SEQ ID NO: 52):EVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_18 (SEQ ID NO: 53):EVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGLTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTLVTVSS TNF_37 (SEQ ID NO: 54):DVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTLVTVSS TNF_66 (SEQ ID NO: 55):HVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGLTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTLVTVSS TNF_68 (SEQ ID NO: 56):HVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGLATHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTLVTVSS TNF_78 (SEQ ID NO: 57):HVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADRKEREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_67 (SEQ ID NO: 58):HVQLVESGGGSVQAGGSLRLSCAASGFRYAAYCMGWFRQADGKVREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTLVTVSS TNF_6 (SEQ ID NO: 59):QVQLVESGGGSVQAGGSLRLSCAASGFIDSFGVMAWFRQAPGKEREGVAAVYRRAGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDSAMYYCAARTYGSVSSWTGYKYWGQGTQVTVSS TNF_7 (SEQ ID NO: 60):DVQLVESGGGSVQAGGSLRLSCAASGFIDSFGVMAWFRQTPGKEREGVAAVYRRAGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDSAMYYCAARTYGSVSSWTGYKYWGQGTQVTVSS TNF_13 (SEQ ID NO: 61):DVQLVESGGGSVQVGGSLTLSCAVSGYTDSYGVMAWFRQAPGKEREGVASIYRNSGITYYPDSVKGRFTISRDNAKNTVLLQMNSLKPEDSATYYCAVRSFGSVSTWAGYVYWGQGTQVTVSS TNF_60 (SEQ ID NO: 62):DVQLVESGGGSVQAGGSLRLSCAASGFIDSFGVMAWFRQAPGKEREGVAAVYRRAGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDSAMYYCAARTYGSVSSWTGYKYWGRGTQVTVSS TNF_73 (SEQ ID NO: 63):DVQLVESGGGSVRAGGSLRLSCTASGDTSKSDCMAWFRQAPGKERERVGAIYTRNGYTHYADSVNGRFTISQDNAKNALYLQMSGLKPEDTAMYYCAARFRIYGQCVEDDDIDYWGQGTLVTVSS TNF_69 (SEQ ID NO: 64):EVQLVESGGGSVLAGGSLRLSCVASGFTSRYNYMAWFRQAPGKEREGVATIGTASGSADYYGSVKDRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAARTYGTISLTPSDYRYWGQGTLVTVSS TNF_76 (SEQ ID NO: 65):QVQVVEYGGGSVQAGETVRLSCTASGFTFAEADMGWYRQAPGHEWELVSNITTEGITSEASSSYADSVRGRFTIFDNAKNMVYLQMNSLKHEDTAVYYCAPDPYAYSTYREYCTWAQGTQGTLVTVSS TNF_62 (SEQ ID NO: 66):QVQLVESGGGPVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCSWAQGTQGTLVTVSS TNF_43 (SEQ ID NO: 67):QVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTLVTVSS TNF_15 (SEQ ID NO: 68):QVQPVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGAQGTLVTVSS TNF_11 (SEQ ID NO: 69):QVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCSWAQGTQGTQVTVSS TNF_17 (SEQ ID NO: 70):QVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTQVTVSS TNF_63 (SEQ ID NO: 71):QVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTLVTVSS TNF_20 (SEQ ID NO: 72):HVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTQVTVSS TNF_58 (SEQ ID NO: 73):EVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGALVTVSS TNF_27 (SEQ ID NO: 74):EVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTLVTVSS TNF_28 (SEQ ID NO: 75):EVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCSWAQGTQGTQVTVSS TNF_4 (SEQ ID NO: 76):EVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTQVTVSS TNF_14 (SEQ ID NO: 77):DVQLVESRGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTLVTVSS TNF_3 (SEQ ID NO: 78):DVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHVCELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCSWAQGTQGTQVTVSS TNF_1 (SEQ ID NO: 79):DVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGLECELVSTITTEGITSEASSYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSEYCTWAQGTQGTLVTVSS TNF_45 (SEQ ID NO: 80):DVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTLVTVSS TNF_ 22 (SEQ ID NO: 81):DVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSVASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTQVTVSS

The in vitro growth inhibition of several TNF-alpha sdAbs was tested, asdescribed below. Additionally, the present invention comprises one ormore mouse monoclonal antibodies which are directed against one or moredomains of the anti-TNF-alpha sdAb of the invention. The mousemonoclonal antibody can be generated by methods that are known by one ofskill in the art, as described above. The mouse monoclonal antibody canbe used in diagnostic assays, such as, for example, an immunoassay suchas an ELISA in order to measure the amount of anti-TNF-alpha sdAbpresent in a patient's serum.

The RAF proteins are a family of serine/threonine-specific kinases thatserve as a central intermediate in transmitting extracellular signals tothe mitogen-activated protein kinase cascade, which controls cellgrowth, differentiation and survival. BRAF is a member of the RAF familythat is activated by members of the Ras family upon growthfactor-induced stimulation. Active Ras can induce heterodimerization ofcRaf and BRAF and this may explain the observed cooperativity of cRafand BRaf in cells responding to growth factor signals. Activatingmutations in the BRAF gene are present in a large percentage of humanmalignant melanomas and in a proportion of colon cancers. The vastmajority of these mutations result in a valine to glutamic acid changeat residue 599 within the activation segment of BRAF.

Anti-BRAF sdAbs were developed to target wild-type and mutated BRAF inorder to disrupt its role in malignant cells such as, for example, cellsinvolved in colon cancer and other malignancies.

Using methods that are well-known in the art, recombinant human BRAFprotein was used to generate sdAbs that are directed against or can bindto an epitope of BRAF.

Additionally, the present invention comprises one or more mousemonoclonal antibodies which are directed against one or more domains ofthe anti-BRAF sdAb of the invention. The mouse monoclonal antibody canbe generated by methods that are known by one of skill in the art. Themouse monoclonal antibody can be used in diagnostic assays, for example,the antibody can be used in an immunoassay such as an ELISA in order tomeasure the amount of anti-BRAF sdAb present in a patient's serum.

Examples Example 1: Anti-STAT3 VHH13 (SEQ ID NO:3) sdAb Binds STAT3

In this example, the affinity of two VHH targets against STAT3 wasmeasured using Octet based label-free binding assay. Anti-STAT3 VHH13(SEQ ID NO:3) sdAb, anti-KRAS (negative control) and GST-STAT3 (16 kDamonovalent antigen, Creative BioMart #STAT3-1476H) were used as antigenprobes in this assay. The GST-STAT3 protein was captured at 20 μg/ml inPBS using aminopropylsilane (APS) dip and read biosensors, specificallymeant for hydrophobic protein. The probes were then dipped into wellswith the GST-STAT3 protein, anti-STAT3 VHH13 (SEQ ID NO:3) sdAb oranti-KRAS at a concentration as indicated. The association rate (onrate) of the antigen was measured. The sensors were quenched with 1% BSAin water. The probes were dipped into assay buffer (PBS) and thedissociation rate (off rate) was measured.

The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (KD) wasdetermined from the obtained affinity constant (KA), and KD using 1:1global fit analysis Fortebio software as shown below in Table 1.Affinity was determined by averaging KD values for curves with R2values >0.95. The 250 nM anti-STAT3 VHH13 data point was omitted as itis an outlier. It was determined that the anti-STAT3 VHH13 (SEQ ID NO:3)sdAb affinity was 1.16×10⁻⁷. The affinity of anti-KRAS VHH was notdetermined.

TABLE 1 Local Fit Analysis, Highlighted Values Used to Determine theAffinity to be 1.16 × 10⁻⁷ Sensor Type Sample ID Loading Sample ID VHHConc. (nM) KD (M) kon (1 Ms) koff(1/s) Full R{circumflex over ( )}2 APS(Aminopropylsilane ANTI-STAT3 VHH13 STAT3 20 μg/ml 1000 1.168E−073.16E+05 3.69E−02 0.985 APS (Aminopropylsilane ANTI-STAT3 VHH13 STAT3 20μg/ml 500 1.012E−07 4.04E+05 4.09E−02 0.974 APS (AminopropylsilaneANTI-STAT3 VHH13 STAT3 20 μg/ml 250  <1.0E−12 4.69E+91 5.11E−02 0.980APS (Aminopropylsilane ANTI-STAT3 VHH13 STAT3 20 μg/ml 125 1.474E−073.09E+05 4.55E−02 0.991 APS (Aminopropylsilane ANTI-STAT3 VHH13 STAT3 20μg/ml 62.5 9.921E−08 2.71E+05 2.69E−02 0.975 APS (AminopropylsilaneANTI-STAT3 VHH13 STAT3 20 μg/ml 31.3  1.53E−06 6.75E+04 1.03E−01 0.656APS (Aminopropylsilane ANTI-kras STAT3 20 μg/ml 1000  6.75E−08 1.19E+048.01E−04 0.917 APS (Aminopropylsilane ANTI-kras STAT3 20 μg/ml 5002.916E−08 1.65E+04 4.80E−04 0.890 APS (Aminopropylsilane ANTI-kras STAT320 μg/ml 250 4.324E−09 8.93E+04 3.86E−04 0.276 APS (AminopropylsilaneANTI-kras STAT3 20 μg/ml 125 NA NA NA NA APS (AminopropylsilaneANTI-kras STAT3 20 μg/ml 62.5 NA NA NA NA APS (AminopropylsilaneANTI-kras STAT3 20 μg/ml 31.3 NA NA NA NA

Example 2: Immunoprecipitation Studies

The specificity of STAT3 sdAbs was assayed in human breast cancer cells.In this example, MDA-MB-231 human breast cancer cells were grown to 50%to 70% confluence. The cells were then disrupted in freshly preparedice-cold lysis buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 0.1% NP-40, 10%glycerol, 1 mM sodium vanadate, 1 mM sodium fluoride, 1 mMdithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin,10 μg/mL leupeptin) for 45 minutes on ice. Lysates were thencentrifuged, the supernatant collected, and protein concentration wasdetermined using a modified Lowry method (Bio Rad, Hercules, Calif.).Total protein (1 mg) was incubated with 1.5 mg of Dynabeads (Invitrogen)with sdAbs against STAT3, a positive control (STAT3, cat#SC-482, SantaCruz Biotechnology, Dallas, Tex.), or negative control (STAT-1,cat#9172, Cell Signaling, Danvers, Mass.) for 1 hour at 4° C. Beads werethen washed. Following the final wash, 60 μl of lysis buffer was added,and the resulting supernatant was subject to Western blot analysis.Briefly, samples were separated on 10% polyacrylamide gels andtransferred to a nitrocellulose membrane. The membranes were blocked,then incubated with appropriate primary and secondary antibodies.Anti-STAT3 antibody, used as a positive control, was from Cell Signaling(Cat#4904, Danvers, Mass.). The chemiluminescence reaction was performedusing the ECL system from Santa Cruz Biotechnology (Dallas, Tex.).

As illustrated in FIG. 3, endogenous STAT3 immunoprecipitated with allsdAbs tested at varying amounts. M is the Marker lane containing themarker, lane 1 contained STAT3 VHH13 (SEQ ID NO:3) produced and isolatedfrom mammalian cells, lane 2 contained STAT3 VHH14 (SEQ ID NO:4)produced and isolated from mammalian cells, lane 3 contained STAT3 VHH13(SEQ ID NO:3) produced and isolated from bacterial cells, lane 4contained STAT3 VHH14 (SEQ ID NO:4) produced and isolated from bacterialcells, lane 5 was the positive STAT3 antibody, lane 6 used STAT-1 as anegative control, showed no band.

Example 3: Anti-STAT3 Bacterial VHH13 Binds with High Affinity to CellLines Contining Constitutively Actived STAT3

The specificity of bacterial anti-STAT3 VHH13 (SEQ ID NO:3) usingconstitutively activated STAT3 in human (PANC-1 and DU145) and murine(4T1) cell lines was assayed. Commercial HeLa cells were also treatedwith interferon gamma (INFF) in order to induce phosphorylated STAT3.The PC-3 STAT3 null cell line was used as a negative control.

The cells were grown to 50% to 70% confluence, then disrupted in freshlyprepared ice-cold lysis buffer as described above for 45 minutes on ice.Lysates were then centrifuged, the supernatant collected, and proteinconcentration was determined as described above. Total protein (1 mg)was incubated with 1.5 mg of Dynabeads (Invitrogen) containing thebacterial anti-STAT3 VHH13 (SEQ ID NO:3) or negative control (KRAS,Creative Biolabs, Shirley, N.Y.) for 1 hour at 4° C. Beads were thenwashed. Following the final wash, 60 μl of lysis buffer was added, andthe resulting supernatant was subject to Western Blot analysis asdescribed in Example 2.

As illustrated in FIG. 4, endogenous STAT3 was immunoprecipitated bybacterial VHH13 STAT3 (SEQ ID NO:3) in the constitutively activatedSTAT3 cell lines: PANC-1 (lane 1), DU145 (lane 2), and 4T1 (lane 4).Furthermore, anti-STAT3 VHH13 (SEQ ID NO:3) sdAb bound to thePhospho-STAT3 in HeLa lysate (lane 3). No bands were noted for eitherPANC-1 KRAS, lane 3, or PC-3 (negative control), lane 6. These resultsindicate that anti-STAT3 VHH13 (SEQ ID NO:3) sdAb can bind bothphosphorylated and un-phosphorylated STAT3.

Example 4: Cytotoxicity Studies of Anti-STAT3 sdAbs in MDA-MB-231 CancerCell Lines

In this example, the anti-proliferative effects of anti-STAT3 sdAbs wereassayed using the human breast cancer cell line MDA-MB-231. For theexperiments, MDA-MB-231 cells were grown until they reached a confluencyof 90%. At that time, cells were washed, trypsinized and counted using aCoulter Counter (Beckman, Brea, Calif.). The proliferation studies werecarried out using the 3-[4,5-dimethylthiaolyl]-2,5-diphenyltetrazoliumbromide (MTT) assay. For this, cells were seeded in a 96-well plate at adensity of 5×10³ per well as indicated by the manufacturer (RocheDiagnostics Corporation, Indianapolis, Ind.). Cells were allowed toadhere for 24 hours and then the sdAbs were added at the appropriateconcentrations (i.e., 0, 0.5, 1.0, 10.0, or 100 μg/ml). Cells werecounted on day 3. For the 5-day treated cells, fresh media containingthe sdAbs was refreshed on day 3. At the time of termination, 10 μl ofMTT reagent (0.5 mg/mL) was added to each well as indicated by themanufacturer. After a 4 hour incubation period, 100 μl of solubilizationsolution was added and the plate was placed in the incubator overnight.All the plates were read at 570 nm using the Biotek plate reader(Winooski, Vt.).

All data were analyzed using GraphPad InStat 3 (GraphPad Software, Inc.,La Jolla, Calif.). Treatments groups were compared with vehicle controlgroup using one-way ANOVA. If a significant difference (p<0.05) wasobserved, the Tukey-Kramer multiple comparison test was conducted.

Based on the MTT experiment, the bacterial VHH13 anti-STAT3 (SEQ IDNO:3) sdAb was found to be effective in inhibiting cell growth at days 3and 5 post-treatment, as shown in Tables 2-5 below.

TABLE 2 Mean Absorbance (570 nM) ± S.E. Day 3 Post Treatment withAnti-STAT3 sdAbs in MDA-MB-231 Cells Treatment Control 0.5 μg/ml 1.0μg/ml 10.0 μg/ml 100 μg/ml p-value* H.VHH13 0.444 ± 0.030 0.504 ± 0.0430.545 ± 0.060 0.603 ± 0.025 0.272 ± 0.011 0.001 H.VHH14 0.404 ± 0.0110.485 ± 0.040 0.402 ± 0.017 0.588 ± 0.020 0.416 ± 0.030 0.002 B.VHH130.550 ± 0.036 0.685 ± 0.018 0.716 ± 0.023 0.355 ± 0.033 0.059 ± 0.001<0.0001 B.VHH14 0.593 ± 0.014 0.666 ± 0.022 0.644 ± 0.045 0.456 ± 0.0480.255 ± 0.005 <0.0001 *One Way Analysis of Variance (ANOVA);Tukey-Kramer Multiple Comparison Test

TABLE 3 Effects of Anti-STAT3 sdAb Treatment on MDA-MB-231 CellProliferation after 3 Days of Treatment Treatment μg/ml % Inhibitionp-value* H.VHH13 0.5 NS 1.0 NS 10.0 NS 100.0 38.7 P < 0.05 H.VHH14 0.5NS 1.0 0.5 NS 10.0 NS 100.0 NS B.VHH13 0.5 NS 1.0 NS 10.0 35.5 P < 0.001100.0 89.3 P < 0.001 B.VHH14 0.5 NS 1.0 NS 10.0 23.1 P < 0.05 100.0 57.0P < 0.001 *One Way Analysis of Variance (ANOVA); Tukey-Kramer MultipleComparison Test

TABLE 4 Mean Absorbance (570 nM) ± S.E. Day 5 Post Treatment withAnti-STAT3 sdAb in MDA-MB-231 Cells Treatment Control 0.5 μg/ml 1.0μg/ml 10.0 μg/ml 100 μg/ml p-value* H.VHH13 1.100 ± 0.088 0.955 ± 0.0130.963 ± 0.018 0.832 ± 0.028 0.721 ± 0.025 0.0012 H.VHH14 0.983 ± 0.0230.890 ± 0.021 0.935 ± 0.037 0.804 ± 0.015 0.797 ± 0.010 0.0007 B.VHH130.804 ± 0.046 0.761 ± 0.055 0.653 ± 0.024 0.506 ± 0.030 0.083 ± 0.005<0.0001 B.VHH14 0.677 ± 0.015 0.733 ± 0.038 0.794 ± 0.023 0.640 ± 0.0110.549 ± 0.023 <0.0001 *One Way Analysis of Variance (ANOVA);Tukey-Kramer Multiple Comparison Test

TABLE 5 Effects of Anti-STAT3 sdAb Treatment on MDA-MB-231 CellProliferation after 5 Days of Treatment Treatment μg/ml % Inhibitionp-value* H.VHH13 0.5 13.2 NS 1.0 12.5 NS 10.0 24.4 P < 0.01 100.0 34.5 P< 0.001 H.VHH14 0.5 9.5 NS 1.0 4.9 NS 10.0 18.2 P < 0.001 100.0 18.9 P <0.001 B.VHH13 0.5 5.4 NS 1.0 18.8 NS 10.0 37.1 P < 0.001 100.0 89.7 P <0.001 B.VHH14 0.5 0 NS 1.0 0 NS 10.0 5.5 NS 100.0 18.9 P < 0.05 *One WayAnalysis of Variance (ANOVA); Tukey-Kramer Multiple Comparison Test

Example 5: Cytotoxicity Studies of Anti-STAT3 sdAbs in Human Breast(MDA-MB-231) and Pancreatic (PANC-1) Cancer Cell Lines

In this Example, the anti-proliferative effects of anti-STAT3 VHH13 (SEQID NO:3) and the VHH14 (SEQ ID NO:4) sdAbs were assayed using the humanbreast cancer cell line MDA-MB-231 and the human pancreatic cancer cellline PANC-1. For the experiments, MDA-MB-231 and PANC-1 cells were grownuntil they were 90% confluent. At that time, cells were washed,trypsinized and counted using a Coulter Counter (Beckman, Brea, Calif.).The proliferation studies were carried out using the MTT assay describedabove. For the 5-day treated cells, fresh media containing theanti-STAT3 sdAbs was refreshed on day 3.

All data were analyzed using GraphPad InStat 3. Treatments groups werecompared with vehicle control group using one-way ANOVA. If asignificant difference (p<0.05) was observed, the Tukey-Kramer multiplecomparison test was conducted.

Based on the MTT experiment, both the VHH13 (SEQ ID NO:3) and the VHH14(SEQ ID NO:4) were found to inhibit cell growth in both the MDA-MB-231and PANC-1 cancer cells, as shown in Tables 6-13 below.

TABLE 6 Mean Absorbance (570 nM) ± S.E. Day 3 Post Treatment With sdAbsin the MDA-MB-231 Cells Treatment Experiment Control 10.0 μg/ml 100μg/ml p-value* B.VHH13 1 0.550 ± 0.036 0.355 ± 0.033 0.059 ± 0.001<0.0001 2 0.735 ± 0.092 0.489 ± 0.019 0.449 ± 0.054 0.0355 3 0.627 ±0.033 0.432 ± 0.060 0.078 ± 0.001 0.0002 4 0.648 ± 0.090 0.576 ± 0.0610.063 ± 0.002 0.0011 Overall Mean 0.640 ± 0.038 0.463 ± 0.047 0.163 ±0.10  0.0019 B.VHH14 1 0.593 ± 0.014 0.456 ± 0.048 0.255 ± 0.005 0.00052 0.624 ± 0.046 0.499 ± 0.018 0.357 ± 0.019 0.0025 3 0.816 ± 0.088 0.502± 0.048 0.308 ± 0.021 0.0026 4 0.729 ± 0.051 0.559 ± 0.041 0.287 ± 0.0210.0007 Overall Mean 0.691 ± 0.051 0.504 ± 0.021 0.302 ± 0.043 <0.0001*One Way Analysis of Variance (ANOVA); Tukey-Kramer Multiple ComparisonTest

TABLE 7 Mean Absorbance (570 nM) ± S.E. Day 5 Post Treatment withAnti-STAT3 sdAbs in MDA-MB-231 Cells Treatment Experiment Control 10.0μg/ml 100 μg/ml p-value* B.VHH13 1 0.804 ± 0.046 0.506 ± 0.030 0.083 ±0.005 <0.0001 2 0.561 ± 0.024 0.417 ± 0.011 0.266 ± 0.015 <0.0001 30.970 ± 0.048 0.814 ± 0.052 0.105 ± 0.005 <0.0001 4 0.757 ± 0.118 0.665± 0.036 0.087 ± 0.004 0.011 Overall Mean 0.773 ± 0.084 0.601 ± 0.0880.135 ± 0.044 0.0005 B.VHH14 1 0.677 ± 0.015 0.640 ± 0.011 0.549 ± 0.0230.0047 2 0.456 ± 0.037 0.338 ± 0.023 0.274 ± 0.032 0.0166 3 0.983 ±0.019 0.930 ± 0.044 0.578 ± 0.039 0.0004 4 1.092 ± 0.053 0.842 ± 0.0520.499 ± 0.036 0.0004 Overall Mean 0.802 ± 0.145 0.688 ± 0.131 0.475 ±0.0690 0.2022 *One Way Analysis of Variance (ANOVA); Tukey-KramerMultiple Comparison Test

TABLE 8 Mean Absorbance (570 nM) ± S.E. Day 3 Post Treatment withAnti-STAT3 sdAbs in the PANC-1 Cells Treatment Experiment Control 10.0μg/ml 100 μg/ml p-value* B.VHH13 1 0.756 ± 0.045 0.432 ± 0.015 0.307 ±0.012 <0.0001 2 1.347 ± 0.189 0.491 ± 0.087 0.169 ± 0.094 0.0019 3 1.025± 0.056 0.493 ± 0.029 0.166 ± 0.028 <0.0001 Overall Mean 1.043 ± 0.1710.472 ± 0.020 0.214 ± 0.047 0.0034 H.VHH13 1 1.541 ± 0.097 1.066 ± 0.1530.732 ± 0.015 0.0046 2 1.611 ± 0.119 1.353 ± 0.119 0.762 ± 0.654 0.35273 1.074 ± 0.040 0.897 ± 0.154 0.700 ± 0.082 0.1092 Overall Mean 1.409 ±0.169 1.105 ± 0.133 0.731 ± 0.181 0.0238 H.VHH14 1 1.195 ± 0.205 0.920 ±0.133 0.808 ± 0.239 0.4161 2 1.423 ± 0.038 1.183 ± 0.114 0.993 ± 0.0880.0338 3 1.293 ± 0.169 1.163 ± 0.044 0.916 ± 0.088 0.1330 Overall Mean1.304 ± 0.066 1.089 ± 0.085 0.906 ± 0.054 0.0188 *One Way Analysis ofVariance (ANOVA); Tukey-Kramer Multiple Comparison Test

TABLE 9 Mean Absorbance (570 nM) ± S.E. Day 5 Post Treatment withAnti-STAT3 sdAbs in PANC-1 Cells Treatment Experiment Control 10.0 μg/ml100 μg/ml p-value* B.VHH13 1 0.687 ± 0.047 0.433 ± 0.036 0.243 ± 0.0240.0004 2 1.670 ± 0.196 0.869 ± 0.053 0.211 ± 0.006 0.0004 3 1.389 ±0.044 0.627 ± 0.073 0.203 ± 0.013 <0.0001 Overall Mean 1.249 ± 0.2920.643 ± 0.126 0.219 ± 0.012 0.0208 H.VHH13 1 1.462 ± 0.150 1.128 ± 0.1050.839 ± 0.117 0.0349 2 1.792 ± 0.202 1.341 ± 0.095 0.911 ± 0.079 0.01133 1.605 ± 0.289 1.161 ± 0.140 0.820 ± 0.005 0.0638 Overall Mean 1.620 ±0.096 1.210 ± 0.066 0.857 ± 0.028 0.0007 H.VHH14 1 1.992 ± 0.105 1.859 ±0.033 0.095 ± 0.003 <0.0001 2 1.517 ± 0.050 1.165 ± 0.015 1.169 ± 0.0500.0015 3 1.579 ± 0.134 1.081 ± 0.103 0.998 ± 0.049 0.0136 Overall Mean1.696 ± 0.149 1.368 ± 0.247 0.754 ± 0.333 0.0967 *One Way Analysis ofVariance (ANOVA); Tukey-Kramer Multiple Comparison Test

TABLE 10 Mean Growth Inhibition Post 3 Days of Anti-STAT3 sdAbsTreatment on MDA-MB-231 Cell Proliferation Treatment ExperimentP-value^(a) 10.0 μg/ml P-value^(b) 100 μg/ml P-value^(b) B.VHH13 1 P <0.0001 35.5 P < 0.001 89.3 P < 0.001 2 P = 0.03 33.5 ns 38.9 P < 0.05 3P = 0.0001 31.1 P < 0.05 87.6 P < 0.001 4 P = 0.0001 11.1 ns 90.3 P <0.01 Overall 27.8 76.5 Average % Inhibition B.VHH14 1 P < 0.001 23.1 P <0.05 57.0 P < 0.001 2 P = 0.03 20.0 ns 42.8 P < 0.01 3 P = 0.03 38.5 P <0.05 62.3 P < 0.01 4 P = 0.006 23.3 ns 60.6 P < 0.001 Overall 26.2 55.7Average % Inhibition ^(a)One-way Analysis of Variance (ANOVA); ^(b)Posttest = Tukey-Kramer Multiple Comparisons Test

TABLE 11 Mean Growth Inhibition Post 5 days of Anti-STAT3 sdAbsTreatment on MDA-MB-231 Cell Proliferation Treatment ExperimentP-value^(a) 10.0 μg/ml P-value^(b) 100 μg/ml P-value^(b) B.VHH13 1 P <0.0001 37.1 P < 0.001 89.7 P < 0.001 2 P < 0.0001 25.7 P < 0.001 52.6 P< 0.001 3 P < 0.0001 16.1 ns 89.2 P < 0.001 4 P = 0.001 12.2 ns 88.5 P <0.01 Overall 22.8 80.0 Average % Inhibition B.VHH14 1 P < 0.0001 5.5 ns18.9 P < 0.05 2 P = 0.02 25.9 ns 39.9 P < 0.05 3 P = 0.0004 5.4 ns 41.2P < 0.001 4 P = 0.0004 22.9 P < 0.05 54.3 P < 0.001 Overall 14.9 38.6Average % Inhibition ^(a)One-way Analysis of Variance (ANOVA); ^(b)Posttest = Tukey-Kramer Multiple Comparisons Test

TABLE 12 Mean Growth Inhibition Post 3 Days of Anti-STAT3 sdAbsTreatment on PANC-1 Cell Proliferation Treatment Experiment P-value^(a)10.0 μg/ml P-value^(b) 100 μg/ml P-value^(b) B.VHH13 1 P < 0.0001 42.9 P< 0.001 59.4 P < 0.001 2 P = 0.03 63.5 P < 0.05 87.5 P < 0.01 3 P <0.0001 51.9 P < 0.001 83.8 P < 0.001 Overall 52.8 76.9 Average %Inhibition H.VHH13 1 P = 0.005 30.8 P < 0.05 52.5 P < 0.01 2 P = 0.00216.0 ns 52.7 P < 0.01 3 P = 0.11 16.5 ns 34.8 ns Overall 21.1 46.7Average % Inhibition H.VHH14 1 P = 0.42 23.0 ns 32.4 ns 2 P = 0.03 16.9ns 30.2 P < 0.05 3 P = 0.13 10.1 ns 29.2 ns Overall 16.7 30.6 Average %Inhibition ^(a)One-way Analysis of Variance (ANOVA); ^(b)Post test =Tukey-Kramer Multiple Comparisons Test

TABLE 13 Mean Growth Inhibition Post 5 Days of Anti-STAT3 sdAbsTreatment on PANC-1 Cell Proliferation Treatment Experiment P-value^(a)10.0 μg/ml P-value^(b) 100 μg/ml P-value^(b) B.VHH13 1 P = 0.0004 37.0 P< 0.01 64.6 P < 0.001 2 P = 0.0004 48.0 P < 0.01 87.4 P < 0.001 3 P <0.0001 54.9 P < 0.001 85.4 P < 0.001 Overall 46.6 79.1 Average %Inhibition H.VHH13 1 P = 0.03 22.8 ns 42.6 P < 0.05 2 P = 0.01 25.2 ns49.2 P < 0.01 3 P = 0.06 27.7 ns 48.9 ns Overall 25.2 46.9 Average %Inhibition H.VHH14 1 P = 0.08 26.8 ns 14.8 ns 2 P = 0.002 23.2 P < 0.0122.9 P < 0.01 3 P = 0.02 31.5 P < 0.05 36.8 P < 0.05 Overall 27.2 24.8Average % Inhibition ^(a)One-way Analysis of Variance (ANOVA); ^(b)Posttest = Tukey-Kramer Multiple Comparisons Test

Example 6: Anti-Proliferative Actions of STAT3 sdAbs in the Human BreastCancer and Human Prostate Cancer Cell Lines

The anti-proliferative effects of the STAT3 VHH13 (SEQ ID NO:3) sdAbwere assayed in the human breast cancer cell line MDA-MB-231 and thehuman prostate cancer cell lines DU145. For the experiments, cancercells were grown until they reached 90% confluence. At that time, cellswere washed, trypsinized, and counted using a Coulter Counter (Beckman,Brea, Calif.). The proliferation studies done using the MTT assay asdescribed above.

The anti-proliferative properties of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb on MDA-MB-231 cells were compared to its actions on DU145cells. As shown in Table 14, MDA-MB-231 cells treated with theanti-STAT3 (SEQ ID NO:3) sdAbs showed an average growth inhibition of29.6 and 91.2 at 50.0 and 100 μg/ml, respectively. In the DU145 cells, asimilar growth inhibition (31.2 and 92.1% for 50.0 and 100 μg/ml,respectively) was seen as set forth in Table 15.

TABLE 14 Anti-proliferative Actions of Anti-STAT3 Bacterial VHH13 sdAbson MDA-MB-231 Breast Cancer Cells Experiment 1 Experiment 2 Experiment 3Average Absorbance Absorbance Absorbance Absorbance (% Inhibition) (%Inhibition) (% Inhibition) (% Inhibition) p-value* control 0.93 1.251.46 1.21  50 μg 0.82 (12.0) 0.99 (20.5) 0.64 (56.2) 0.82 (32.6) NS 100μg 0.07 (93.1) 0.12 (90.1) 0.14 (90.5) 0.11 (91.0) <0.001 *One WayAnalysis of Variance (ANOVA); Tukey-Kramer Multiple Comparison Test

TABLE 15 Anti-proliferative Actions of Anti-STAT3 Bacterial VHH13 sdAbson DU145 Prostate Cancer Cells Experiment 1 Experiment 2 Experiment 3Average Absorbance Absorbance Absorbance Absorbance (% Inhibition) (%Inhibition) (% Inhibition) (% Inhibition) p-value* control 1.05 1.581.61 1.41  50 μg 0.68 (35.7) 1.2 (55.5) 1.03 (35.8) 0.98 (30.5) NS 100μg 0.13 (87.4) 0.12 (95.7) 0.06 (96.1) 0.10 (92.7) <0.001 *One WayAnalysis of Variance (ANOVA); Tukey-Kramer Multiple Comparison Test

Example 7: Anti-Proliferative Effects of STAT3 VHH13 (SEQ ID NO:3) sdAbson Human Cancer Cell Lines

To test the anti-proliferative effects of the STAT3 VHH13 (SEQ ID NO:3)sdAbs using the human cancer cell lines: MDA-MB-231, MDA-MB-468, MCF-7,BT474, and DU145 as shown in Table 16.

All human cancer cell lines were obtained from American Type CultureCollection (Manassas, Va.). Cell lines were maintained and cultured inRPMI 1640 media (MDA-MB-231, MDA-MB-468, MCF-7, BT474) or MEM-E (DU145)containing 10% fetal bovine serum, 2 mM L-glutamine and 1%antibiotic-antimycotic solution (10 units/mL penicillin, 10 μg/mLstreptomycin and 25 μg/mL amphotericin B). Cells were kept at 37° C. ina humidified atmosphere of 5% CO₂. Cell culture supplies were obtainedfrom Life Technologies, Inc., (Grand Island, N.Y.). The MTT reagent waspurchased from Sigma Aldrich (St. Louis, Mo.).

For the experiments, cancer cells were grown until they reached 90%confluency. At that time, cells were washed, trypsinized and countedusing a Coulter Counter (Beckman, Brea, Calif.). The proliferationstudies were carried out using the MTT assay as described above.

The anti-proliferative properties of Anti-STAT3 Bacterial VHH13 (SEQ IDNO:3) sdAbs were evaluated on five breast cancer cells of representingvarious classifications (Table 16). As shown in Table 17, all cell linesat 72 hours post treatment showed significant growth inhibition. Thegreatest growth inhibition was noted at 100 and 200 μg/ml dose for allcell lines. The half maximal inhibitory concentration (IC₅₀) for growthin the cell lines tested were: 10.1±2.4, 12.36±1.5, 14.8±1.6, and25.2±14.7 for the MDA-MB-231, MDA-MB-468, MCF-7, and BT474 cell lines,respectively. These data suggest that the triple negative breast cancercell lines require the lowest concentration of VHH13 (SEQ ID NO:3) sdAbsto achieve the IC₅₀ as compared to estrogen/progesterone positive celllines (i.e., MCF-7) or HER2 amplified cell lines (i.e., BT474).

TABLE 16 Breast Cancer Cell Line Characteristics Cell line DiseaseImmunoprofile Classification MDA-MB-231 adenocarcinoma ER⁻, PR⁻, HER2⁻Basal; Claudin-low MDA-MB-468 adenocarcinoma ER⁻, PR⁻, Her2⁻ BasalMDA-MB-453 metastatic ER, PR, HER2⁻ Unclassified carcinoma BT474 ductalcarcinoma Her2 amplified Luminal B MCF-7 adenocarcinoma ER⁺, PR⁺, HER2⁺Luminal A

TABLE 17 Inhibition of Breast Cancer Cell Lines by Anti-STAT3 VHH13 (SEQID NO: 3) sdAbs Cell Line Treatment (μg/ml) Mean Abs % Inhibitionp-value BT474 0 0.634 0.39 0.322 49.3 P < 0.001 0.78 0.462 27.2 P <0.001 1.56 0.502 20.8 P < 0.01 3.13 0.446 29.7 P < 0.001 6.25 0.469 26.1P < 0.001 12.5 0.363 42.7 P < 0.001 25 0.256 59.6 P < 0.001 50 0.14577.2 P < 0.001 100 0.046 92.8 P < 0.001 200 0.040 93.8 P < 0.001 MCF-7 00.590 0.39 0.818 0 0.78 0.785 0 1.56 0.823 0 3.13 0.689 0 6.25 0.43522.1 NS 12.5 0.327 41.6 P < 0.01 25 0.212 62.1 P < 0.001 50 0.057 89.9 P< 0.001 100 0.038 93.2 P < 0.001 200 0.040 92.9 P < 0.001 MDA-MB-468 00.253 0.39 0.311 0 0.78 0.289 0 1.56 0.201 20.6 3.13 0.223 11.9 6.250.230 9.1 12.5 0.130 48.6 P < 0.001 25 0.067 73.5 P < 0.001 50 0.04283.4 P < 0.001 100 0.038 85.0 P < 0.001 200 0.040 84.4 P < 0.001MDA-MB-231 0 0.502 0.39 0.603 0 0.78 0.576 0 1.56 0.570 0 3.13 0.44511.4 P < 0.001 6.25 0.312 37.8 P < 0.001 12.5 0.224 55.4 P < 0.001 250.196 60.9 P < 0.001 50 0.130 74.2 P < 0.001 100 0.041 91.8 P < 0.001200 0.042 91.7 P < 0.001

The actions of anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb was alsoevaluated in the human prostate cancer cell line DU145, as shown inTable 18. The anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb showeddose-dependent growth inhibition in all cancer cells tested.

TABLE 18 Effect of Anti-STAT3 VHH13 sdAbs on Prostate Cancer Cell LinesTreatment (mg/ml) Mean Abs % Inhibition p-value DU145 0 0.771 DU145 0.390.906 0 DU145 0.78 1.023 0 DU145 1.56 0.967 0 DU145 3.13 0.783 0 DU1456.25 0.770 0 DU145 12.5 0.560 27.4 P < 0.05 DU145 25 0.359 53.5 P <0.001 DU145 50 0.161 79.1 P < 0.001 DU145 100 0.039 95.0 P < 0.001 DU145200 0.039 95.0 P < 0.001

Example 8: Maximum Tolerated Dose of Anti-STAT3 Bacterial VHH13 (SEQ IDNO:3) in BALB/C Mice

In this Example, the tolerance of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb was assayed in test animals using the human breast cancercell line MDA-MB-231. For the experiment, a total of 9 BALB/C nudefemale mice (6 to 7 weeks old) were divided into three groups accordingto body weights. (Table 19) Mice (n=3) received either vehicle (PBS) oranti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb at 250 or 500 μg/kg bodyweight/day for five days. During the study, mortality/morbidity wasperformed twice daily. Body weights were recorded on days 1, 4, and 6 ofthe study as well as on the day of study termination (Day 13). Toxicitywas assessed by body weight measurements and mouse behavior compared tovehicle control mice. Upon completion of treatment phase, animals werefollowed for an additional week to note any abnormalities in bodyweights and/or general health post treatment.

TABLE 19 Experimental Design of Maximum Tolerated Dose Study Group #Mice Treatment Dose Route Frequency 1 3 PBS Vehicle — IP 5 days 2 3Bacterial VHH13 250 μg/kg b.w. IP 5 days 3 3 Bacterial VHH13 500 μg/kgb.w. IP 5 days

As illustrated in Table 20, there was no significant difference in bodyweights among the groups, and anti-STAT3 bacterial VHH13 (SEQ ID NO:3)sdAb was not associated with any drug-related deaths at either dosinglevel. Additionally, no behavior changes were observed in the animalstreated with anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb as comparedto the control mice.

TABLE 20 Mean body weights ± S.E Group Randomization Day 1 Day 4 Day 6Day 13 Vehicle 17.1 ± 0.06 17.1 ± 0.07 17.8 ± 0.12 18.1 ± 0.09 18.8 ±0.20 250 μg/kg 17.1 ± 0.06 17.2 ± 0.03 17.2 ± 0.15 17.5 ± 0.15 18.1 ±0.21 500 μg/kg 17.1 ± 0.17 17.1 ± 0.09 17.8 ± 0.18 18.0 ± 0.20 18.5 ±0.18 p-value* >0.9999 0.52 0.05 0.07 0.11 *One Way Analysis of Variance(ANOVA); Tukey-Kramer Multiple Comparison Test

Example 9: Activity of Bacterial Anti-STAT3 VHH13 (SEQ ID NO:3) in NudeBALB/C Mice Xenograft and Human Breast Cancer and Human PancreaticCancer Cells

In this example, the activity of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb was evaluated in mice using the MDA-MB-231 human breastcancer xenograft model and the PANC-1 human pancreatic cancer xenograftmodel. Dosing schedules were as follows: Group 1 (n=6; PBS; IP) dailyfor 14 days [QD×14]; and Group 2 (n-12; 500 μg/kg bw; IP), every day for14 days [QD×14]. An observation period of 5 days followed the drugadministration.

The human breast cancer cell lines (MDA-MB-231 and PANC-1) were obtainedfrom American Type Culture Collection (ATCC) (Manassas, Va.). TheMDA-MB-231 cells were growth in MEM (Life Technologies, Grand Island,N.Y.) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch,Ga.) and Penicillin-Streptomycin-Glutamine (Life Technologies, GrandIsland, N.Y.). The PANC-1 cells were grown in RPMI 1640 media (LifeTechnologies, Grand Island, N.Y.) supplemented with 10% FBS andPenicillin-Streptomycin-Glutamine. All cells were grown in the presenceof 5% CO₂ at 37° C. in an incubator.

Athymic nude-Foxn1^(nu) male mice aged 4 to 5 weeks were purchased fromHarlan Laboratories (Indianapolis, Ind.). Animals were quarantined forone week and housed five mice per cage, with a 12-hr light-dark cycle,and a relative humidity of 50%. Drinking water and diet were supplied tothe animals ad libitum. All animals were housed under pathogen-freeconditions and experiments were performed in accordance with the IITResearch Institute Animal Use and Care Committee. For the MDA-MB-231xenograft study, cells (4×10⁶) in a 100-μL final volume of MEM mediawere injected subcutaneously into right flanks of mice. For the PANC-1xenograft study, cells (5×10⁶) in a 100-μL final volume of RPMI mediawere injected subcutaneously into right flanks of mice. Tumormeasurements for both models were initiated as soon as the tumors werepalpable. Thereafter, tumors were measured twice weekly. Animals wererandomized when tumors reach a range size of 75 to 175 mm³, control(n=6) and a treatment (n=12) groups were randomized using the stratifiedrandom sampling algorithm. Treatment (anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb) or Vehicle (PBS) was initiated the day followingrandomization. The treatment was well tolerated and associated with nodrug-related deaths. No significant body weight loss was noted.

For the MDA-MB-231 xenograft study, the randomization Mean (±SE) tumorsize was: 103.01±11.89 and 102. 61±9.60 for control and treatment groupsrespectively. Mean body weights (±SE) at randomization were: 32.08±0.76and 30.27+0.75 for Group 1 and Group 2, respectively. Table 21 shows themean body weights (±SE) for the entire study.

TABLE 21 Mean body weights ± S.E. Treatment Day 1 Day 6 Day 9 Day 12 Day16 Day 20 Vehicle 31.0 ± 0.83 32.1 ± 0.76 31.9 ± 0.66 32.1 ± 0.68 32.0 ±0.71 32.5 ± 0.88 Anti-STAT3 29.2 ± 0.71 30.3 ± 0.75 30.4 ± 0.79 29.9 ±0.72 30.6 ± 0.74 30.6 ± 0.77 VHH13 p-value* 0.16 0.18 0.27 .09 0.28 0.17*Two-tail T-Test

On day 14 of dosing, the mean tumor size (±SE) for the control was179.11±19.39 versus 118.86±15.94 for treatment group. Mean body weights(±SE) at termination were: 31.98±0.71 and 30.55±0.74 for Group 1 andGroup 2, respectively. Table 22 summarizes the tumor volumes (±SE) forentire study. The % mean tumor growth inhibition in the treatment groupwas 33.64%. The tumor doubling times were as follows: Group 1: 44.27days; and Group 2: 61.06 days. FIG. 5 illustrates the growth inhibitionof anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb in the MDA-MB-231xenograft model. Anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb showedsignificant growth inhibition (p=0.047). Thus, anti-STAT3 bacterialVHH13 (SEQ ID NO:3) sdAb has chemotherapeutic activity in the MDA-MB-231human breast cancer model system.

TABLE 22 Individual Tumor Measurements (mm³) for the MDA-MB-231Xenograft Model Group Animal # Day 1 Day 6 Day 9 Day 12 Day 16 Day 20 11 117.43 141.72 135.00 139.31 127.93 133.19 2 130.30 142.83 206.15256.99 244.06 243.00 3 78.00 105.97 114.04 144.06 154.50 158.94 4 118.24162.41 171.39 225.59 181.32 217.97 5 71.10 109.03 133.13 168.80 187.73164.45 Mean 103.01 132.39 151.94 186.95 179.11 183.51 S.E. 11.89 10.8216.42 23.28 19.39 20.28 2 6 123.94 114.91 129.22 176.04 170.09 162.98 785.93 101.06 112.60 112.24 139.56 96.43 8 147.34 148.72 169.69 185.08170.07 256.71 9 115.91 103.64 108.37 141.21 144.51 119.42 10 73.23 82.59110.13 91.22 166.77 285.88 11 163.73 178.23 183.79 165.52 214.28 129.5112 75.54 83.94 103.68 119.88 104.26 99.48 13 70.04 89.24 102.60 75.2557.65 95.23 14 101.62 65.09 82.02 68.01 61.41 61.83 15 67.83 62.21 59.0077.04 65.49 82.73 16 131.93 75.28 76.21 53.55 73.66 51.61 17 74.28109.06 111.92 89.94 58.56 100.07 Mean 102.61 101.16 112.44 112.92 118.86128.49 S.E. 9.6 9.8 10.3 12.9 15.9 21.1 P-value 0.98 0.08 0.06 0.01 0.050.14

For the PANC-1 xenograft study, the randomization Mean (+SE) tumor sizeswere 107.01±4.54 in the control and 110. 58±6.18 in the treatmentgroups. Mean body weights (±SE) at randomization were: 29.0±0.81 and28.5±0.70 for Group 1 and Group 2, respectively. Mean body weights (±SE)at termination were: 31.2±0.99 and 30.1±0.75 for Group 1 and Group 2,respectively. Table 23 summarizes the mean body weights (±SE) for entirestudy. On day 14 of dosing, the mean tumor size (±SE) for control was287.30±33.94 versus 318.74+29.76 for treatment group. Table 24summarizes the tumor volumes (±SE) for entire study.

TABLE 23 Mean body weights ± S.E. Treatment 2/19 2/24 2/27 3/2 3/6 3/10Vehicle Control 31.0 ± 0.83 32.1 ± 0.76 31.9 ± 0.66 32.1 ± 0.68 32.0 ±0.71 32.5 ± 0.88 Anti-STAT3 29.2 ± 0.71 30.3 ± 0.75 30.4 ± 0.79 29.9 ±0.72 30.6 ± 0.74 30.6 ± 0.77

The tumor doubling times were as follows: Group 1: 22.44 days, and Group23.02 days. Anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb showed nosignificant growth inhibition in the PANC-1 human pancreatic cancermodel system.

TABLE 24 Individual Tumor Measurements (mm³) for the PANC-1 xenograftModel Group Animal # 2/19 2/24 2/27 3/2 3/6 3/10 1 1 99.77 117.96 134.67161.27 160.79 195.58 2 117.54 137.14 221.14 241.27 303.70 321.45 3120.30 210.99 276.05 322.17 394.96 732.07 4 111.65 135.91 215.87 340.97334.08 382.06 5 90.88 96.35 165.26 156.28 223.17 314.97 6 107.05 156.56192.98 324.34 307.13 573.99 Mean 107.87 142.49 201.00 257.72 287.30420.02 S.E. 11.11 16.01 20.00 34.35 33.94 80.34 2 7 96.31 193.71 275.06317.53 395.37 540.66 8 89.24 90.03 112.43 125.51 189.63 235.08 9 80.62148.97 196.38 187.24 299.84 530.46 10 108.03 144.14 234.46 240.39 288.75421.61 11 77.66 116.21 313.19 290.38 411.66 197.67 12 129.68 143.20290.67 224.92 261.44 343.04 13 108.99 182.30 239.00 254.64 342.19 464.0014 123.27 171.03 223.34 226.88 248.69 324.30 15 144.53 136.03 198.47226.04 247.97 273.58 16 120.96 136.48 226.43 338.06 564.71 883.81 17112.69 144.76 167.12 225.70 223.06 326.19 18 134.95 189.64 193.14 248.01351.63 364.44 Mean 110.58 149.71 222.47 242.11 318.74 408.74 S.E. 6.188.79 15.90 16.30 29.76 53.25 P-value 0.78 0.67 0.43 0.64 0.53 0.91

Example 10: MDA-MB-231 Xenograft Study

In this Example, the efficacy of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb in the MDA-MB-231 human breast xenograft model was furtherevaluated. The dosing schedules were as follows: Group 1 (n=4; PBS; IP)twice a day for 14 days [BID×14]; Group 2 (n=4; 1 mg/kg bw; IP), twice aday for 14 days [BID×14]; Group 3 (n=4; 2 mg/kg bw; IP) twice a day for14 days [BID×14]; and Group 4 (n=4; 2 mg/kg bw; IP) once a day for 14days [QD×14]. An observation period of 7 days followed administration.

The human breast cancer cell lines MDA-MB-231 and athymicnude-Foxn1^(nu) female mice were described above.

MDA-MB-231 cells at a density of 5×10⁶ were injected subcutaneously intothe right flank of the mice at a final volume of 100-μL in MEM media.Tumor measurements were initiated as soon as the tumors were palpable.Thereafter, tumors were measured twice weekly. Animals are randomizedwhen tumors reach a range size of 55 to 150 mm³ using the stratifiedrandom sampling algorithm. Treatment (anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb) or Vehicle (PBS) was initiated the day followingrandomization.

The randomization Mean (±SE) tumor size was: 92.08±13.24, 82.38±5.17,77.47±7.17, and 104.71±14.64 for Groups 1, 2, 3, and 4 respectively. Asshown in Table 25, mean body weights (±SE) at randomization were:23.65±0.72, 23.45±0.66, 23.10±0.20, and 22.45±1.25 for Groups 1, 2, 3,and 4, respectively.

As shown in Table 26, at day 14 of dosing, the mean tumor size (±SE) forcontrol group was 221.51±57.32 versus 67.12±10.66, 58.27±22.54, and131.44±22.86, for treatment group 2, 3, and 4, respectively. At the timeof termination (day 42) mean tumor size (±S.E.) was: 255.42±65.46,55.98±6.94, 41.15±13.21, and 145.51±52.32, for groups 1, 2, 3, and 4,respectively. Mean body weights (±SE) at termination were: 24.80±0.49,23.25±1.20, 24.00±0.32, and 23.2±1.46 for Groups 1, 2, 3, and 4,respectively. The max mean % net weight loss (day) was: 0.7 (36), 1.5(23), 1.8 (36), and 2.2 (29) for Groups 1, 2, 3, and 4, respectively.

Also as shown in Table 26, the mean growth inhibition in the treatmentgroups was 78.3, 75.2, and 55.9, for Groups 2, 3, and 4, respectively.The tumor doubling times were: Group 1: 20.56 days; Group 2: 34.54 days;Group 3: 30.07 days; and Group 4: 27.17 days. There was a growth delayof 13.99, 9.52, and 6.61 days for Groups 2, 3 and 4, respectively. The %treatment/control values for treatment groups were: Group 2: −33.75(tumor stasis); Group 3: −54.4 (tumor regression); and Group 4: 10.28(tumor inhibition). FIG. 6 illustrates the growth inhibition ofanti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb in the MDA-MB-231xenograft model.

Administration of anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb wasassociated with a significant growth inhibition in Group 2 (p=0.02) [1mg/kg; BID×14] and Group 3 (p=0.02) [2 mg/kg; BID×14]. Furthermore,three out of four tumors showed significant regression. Based on thesedata, it is concluded that anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAbhas chemotherapeutic activity in the MDA-MB-231 human breast cancermodel system.

TABLE 25 Mean Body Weights ± S.E. Date/Study Day Dosing Recovery 6/236/26 6/29 7/2 7/6 7/9 7/15 Group Schedule 20 23 26 29 33 36 42 1 PBS;BID x14 23.65 ± 0.72 23.85 ± 0.60 24.18 ± 0.67 24.05 ± 0.63 24.30 ± 0.6724.13 ± 0.72 24.80 ± 0.49 2 1 mg/kg; BID x 14 23.45 ± 0.66 23.10 ± 0.6823.13 ± 0.74 23.13 ± 0.95 23.08 ± 1.01 23.13 ± 1.09 23.25 ± 1.20 3 2mg/kg; BID x 14 23.10 ± 0.20 23.10 ± 0.14 23.20 ± 0.07 23.85 ± 0.3923.80 ± 0.24 23.38 ± 0.23 24.00 ± 0.32 4 2 mg/kg; QD x 14 22.45 ± 1.2522.35 ± 1.32 22.58 ± 1.46 22.08 ± 1.44 22.73 ± 1.47 22.55 ± 1.46 23.20 ±1.38

TABLE 26 Individual Tumor Measurements (mm³) for the MDA-MB-231Xenograft Model Animal Jun. 23, 2015 Jun. 26, 2015 Jun. 29, 2015 Jul. 2,2015 Jul. 6, 2015 Jul. 9, 2015 Jul. 15, 2015 # (20) (23) (26) (29) (33)(36) (42) Group 1 001 93.38 119.07 159.80 197.91 210.95 243.31 265.61002 116.07 241.31 313.16 339.13 362.30 390.48 426.32 003 55.67 83.4598.22 135.50 198.19 204.96 218.29 004 104.82 112.09 118.44 111.07 114.61115.31 111.45 Mean Absolute 92.49 138.98 172.41 195.90 221.51 238.51255.42 Mean Relative 100.00% 150.27% 186.41% 211.82% 239.51% 257.89%276.17% S.E. Mean 13.12 34.97 48.64 51.12 51.56 57.32 65.46 % InhibitionMean Median Absolute 99.10 115.58 139.12 166.71 204.57 224.13 241.95Median Relative 100.00% 116.62% 140.38% 168.22% 206.42% 226.16% 244.14%S.E. Median 13.66 37.49 52.30 53.83 52.48 57.91 65.92 % InhibitionMedian Group 2 005 73.15 54.54 59.17 57.21 56.20 37.13 39.17 006 80.1176.56 80.34 88.75 99.09 87.42 72.18 007 97.22 79.99 78.44 59.90 55.9053.66 60.35 008 81.21 53.58 54.34 67.43 57.30 29.02 52.23 Mean Absolute82.92 66.17 68.07 68.32 67.12 51.81 55.98 Mean Relative 100.00% 79.79%82.09% 82.39% 80.95% 62.48% 67.51% S.E. Mean 5.09 7.03 6.62 7.14 10.6612.93 6.94 % Inhibition Mean 10.34% 52.39% 60.52% 65.12% 69.70% 78.28%78.08% Median Absolute 80.66 65.55 68.80 63.66 56.75 45.40 56.29 MedianRelative 100.00% 81.27% 85.30% 78.93% 70.36% 56.28% 69.79% S.E. Median5.25 7.04 6.63 7.63 12.23 13.45 6.94 % Inhibition Median 18.61% 43.28%50.54% 61.81% 72.26% 79.75% 76.74% Group 3 009 56.41 43.61 33.13 31.7634.11 50.33 18.94 010 84.06 85.18 61.75 80.69 110.72 89.11 73.89 01182.87 54.78 34.92 54.38 78.47 78.68 51.30 012 86.73 44.01 23.09 16.999.78 18.71 20.48 Mean Absolute 77.52 56.89 38.22 45.95 58.27 59.21 41.15Mean Relative 100.00% 73.39% 49.31% 59.28% 75.17% 76.38% 53.09% S.E.Mean 7.08 9.78 8.26 13.90 22.54 15.79 13.21 % Inhibition Mean 16.19%59.06% 77.83% 76.54% 73.69% 75.18% 83.89% Median Absolute 83.46 49.3934.02 43.07 56.29 64.51 35.89 Median Relative 100.00% 59.18% 40.76%51.60% 67.44% 77.29% 43.00% S.E. Median 7.87 10.69 8.61 14.00 22.5616.08 13.56 % Inhibition Median 15.78% 57.27% 75.54% 74.17% 72.49%71.22% 85.17% Group 4 013 88.56 108.35 105.80 102.94 183.39 159.78291.06 014 78.73 51.51 54.20 70.39 84.29 55.83 42.03 015 113.20 85.2969.30 103.16 103.20 87.15 130.64 016 141.91 130.82 87.49 145.68 154.89117.63 118.31 Mean Absolute 105.60 93.99 79.20 105.54 131.44 105.10145.51 Mean Relative 100.00% 89.01% 75.00% 99.94% 124.47% 99.52% 137.79%S.E. Mean 14.11 16.94 11.18 15.44 22.86 22.17 52.32 % Inhibition Mean−14.18% 32.37% 54.06% 46.13% 40.66% 55.94% 43.03% Median Absolute 100.8896.82 78.40 103.05 129.05 102.39 124.47 Median Relative 100.00% 95.98%77.71% 102.15% 127.92% 101.49% 123.38% S.E. Median 14.37 17.02 11.1915.50 22.90 22.22 53.72 % Inhibition Median −1.80% 16.23% 43.65% 38.19%36.92% 54.32% 48.55%

Example 11: Efficacy of Anti-STAT3 Bacterial VHH13 (SEQ ID NO:3) sdAb onThree Human Cancer Xenograft Models

In this Example, the efficacy of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb was evaluated in the MDA-MB-231 Human Breast, PANC-1Pancreatic, and DU145 Prostate cancer xenograft models.

Athymic Nude-Foxn1^(nu) mice, MDA-MB-231 breast cancer cells, PANC-1pancreatic cancer and the DU145 prostate cancer cell lines weredescribed above. The body weight of the mice ranged from 17 to 19 g (34females) and 21 to 23 g (16 males) on Day 1 of the study.

Cells in early passages (4 to 10) were used for implantation into themice and were harvested during log phase growth. MDA-MB-231 (5×10⁶),DU145 (5×10⁶), and PANC-1 (1.5×10⁶) were injected subcutaneously intothe right flank of the mice at a final volume of 100-μL of media. Tumormeasurements were initiated as soon as the tumors were palpable.Thereafter, tumors were measured twice weekly.

Animals were randomized using the stratified random sampling algorithmwhen tumors reach a range size of: 74-120 mm³ (MDA-MB-231), 89-146 mm³(DU145), or 60-160 mm³ (PANC-1). Treatment (containing anti-STAT3bacterial VHH13 (SEQ ID NO:3) sdAb and referred to herein as SBT-100) orVehicle (PBS) was initiated the day following randomization, referred toas day 1.

Anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb was supplied as apre-formulated solution at a concentration of 0.651 mg/ml and was storedat −20° C. until ready to use. The stock solution was diluted in sterilePBS pH 7.6 to provide a 5 mg/kg in a dosing volume of 10 mL/kg. Theworking solution was prepared every 7 days, aliquoted onto seven vialsand stored at 4° C. On each day of treatment, only the needed vial wasbrought to room temperature. All leftover sdAb material was retained at4° C. as need for the next dose. At day 8, any remaining sdAb materialwas discarded and a fresh batch prepared.

Two groups (control and SBT-100) of mice per tumor model were dosedaccording to the protocol shown in Table 27. Dosing schedules were asfollows: Group 1 (n=4; PBS) twice a day for 14 days [BID×14]; Group 2(n=4; SBT-100, 5 mg/kg bw), twice a day for 14 days [BID×14]. Both thevehicle (PBS pH 7.6) and SBT100 were administered intraperitoneally(i.p.) twice a day, six hours apart for fourteen days. Dosing wasconducted according to individual animal weights. A recovery period of 7days followed administration.

TABLE 27 Experimental Design of Xenograft Study # of cells inoculated/ #Dose Model mouse Group Mice Agent (mg/kg) Route Schedule MDA-MB-231 5 ×10⁸ 1 4 Control (PBS) 0 IP BIDx14 2 4 SBT-100 5 IP BIDx14 PANC-1 1.5 ×10⁸  1 4 Control (PBS) 0 IP BIDx14 2 4 SBT-100 5 IP BIDx14 DU145 5 × 10⁸1 4 Control (PBS) 0 IP BIDx14 2 4 SBT-100 5 IP BIDx14

Study Log Study Director Animal Study Management Software

Study Log Study Director Animal Study Management Software (SanFrancisco, Calif.) was used to randomize animals, collect data (e.g.,dosing, body weights, tumors measurements, clinical observations), andconduct data analyses.

In the MDA-MB-231 tumor xenograft model, animals were randomized on day23 post-inoculation with a mean (±SE) tumor size of: 77.98±21.58 and84.71±5.56 for Groups 1 and 2, respectively. Mean body weights (±SE) atrandomization were: 20.04±0.62 and 23.7±1.84 for Groups 1 and 2,respectively. Table 28 summarizes the mean body weights (±SE) for entirestudy. At last day of dosing (Day 14), the mean tumor size (±SE) forcontrol group was 168.28±51.57 versus 83.81±22.65 for SBT-100 treatedmice. Table 29 summarizes the tumor volumes (±SE) for entire study. Atthe time of termination (day 28) mean tumor size (±S.E.) was:270.49±112.35 and 91.72±33.17, for Groups 1 and 2, respectively. Meanbody weights (±SE) at termination were: 25.36±1.07 and 24.25±1.68 forGroups 1 and 2, respectively. At the end of the study, the mean tumorgrowth inhibition in the SBT-100 treated group was 85.8% (p=0.006). FIG.7 illustrates the mean tumor volume. The tumor doubling times were 25.78days versus 111.6 days for Group 1 and Group 2, respectively. The %treatment/control for Group 2 was 13.35 (tumor inhibition).

TABLE 28 Mean Body Weights for Mice in MDA-MB-231 Phase Animal DosingRecovery Group ID 8/28 9/1 9/4 9/8 9/11 9/15 9/18 Control 001 23.4024.80 24.60 25.00 24.70 23.30 25.10 Control 002 22.40 22.50 22.60 22.7022.80 20.60 23.10 Control 003 23.70 24.80 25.20 24.80 24.30 23.30 25.20Control 004 23.70 24.70 25.10 25.30 24.90 22.90 25.40 Mean 23.30 24.2024.38 24.45 24.18 22.53 24.70 Median 23.55 24.75 24.85 24.90 24.50 23.1025.15 SD 0.62 1.13 1.21 1.18 0.95 1.30 1.07 % Change 0.00 3.82 4.56 4.893.73 −3.38 5.97 SBT-100 005 21.70 21.70 21.70 22.40 22.60 21.40 22.20SBT-100 006 25.00 24.30 24.30 24.70 25.30 24.40 25.00 SBT-100 007 22.6023.00 23.10 23.10 23.80 22.80 23.70 SBT-100 008 25.50 25.30 25.50 26.1025.80 25.60 26.10 Mean 23.7 23.575 23.65 24.075 24.375 23.55 24.25Median 23.8 23.65 23.7 23.9 24.55 23.6 24.35 SD 1.84 1.56 1.63 1.66 1.461.84 1.68 % Change 0.00 −0.45 −0.15 1.65 2.96 −0.63 2.38

TABLE 29 Tumor Volumes for MDA-MB-231 Phase Animal Pre-Dosing DosingRecovery Group ID 8/21 8/24 8/27 8/28 9/1 9/4 9/8 9/11 9/15 9/18 Control001 51.00 55.80 80.94 76.35 83.66 94.11 110.78 129.99 162.81 184.15Control 002 75.19 77.22 121.13 120.73 145.12 179.21 203.15 234.05 308.70428.44 Control 003 57.04 57.81 75.32 81.06 93.25 114.27 181.87 242.88295.93 408.67 Control 004 42.92 51.67 106.54 92.23 116.96 142.60 191.58213.48 286.91 303.19 Mean 56.54 60.63 95.98 92.59 109.75 132.55 171.84205.10 263.59 331.11 Median 54.02 56.80 93.74 86.64 105.10 128.44 186.72223.76 291.42 355.93 SD 13.71 11.36 21.58 19.91 27.42 36.92 41.63 51.5767.78 112.35 SBT-100 005 72.25 64.45 80.02 74.07 56.81 49.44 68.70 73.0493.32 116.07 SBT-100 006 61.50 63.08 80.67 79.60 71.92 67.08 87.54115.80 116.97 120.44 SBT-100 007 37.41 35.15 91.93 91.85 50.02 50.3246.10 63.85 66.57 80.57 SBT-100 008 43.80 56.95 86.22 79.94 59.23 60.1954.10 82.57 79.47 49.78 Mean 53.74 54.91 84.71 81.37 59.49 56.76 64.1183.81 89.08 91.72 Median 52.65 60.02 83.45 79.77 58.02 55.25 61.40 77.8186.40 98.32 SD 16.00 13.57 5.56 7.49 9.16 8.43 18.21 22.65 21.56 33.17 %T/C 0.0 32.3 84.0 81.6 13.6 5.9 9.2 20.7 17.6 13.4 p-value 0.800 0.5420.351 0.332 0.013 0.007 0.003 0.005 0.003 0.006

In the DU145 tumor xenograft model, animals were randomized on day 17post-inoculation with a mean (±SE) tumor size of: 111.87±20.53 and111.23±25.16 for Groups 1 and 2, respectively. Mean body weights (±SE)at randomization were: 29.10±1.94 and 30.68±1.56 for Groups 1 and 2,respectively. Table 30 summarizes the mean body weights (±SE) for entirestudy. At last day of dosing (Day 14), the mean tumor size (±SE) forcontrol group was 621.81±276.25 versus 364.14±51.64 for SBT-100 treatedmice. Table 31 summarizes the tumor volumes (±SE) for entire study. Atthe time of termination (day 28) mean tumor size (±S.E.) was:819.42±351.88 and 601.83±131.51, for Groups 1 and 2, respectively. Meanbody weights (±SE) at termination were: 29.20±2.33 and 29.60±1.04 forGroups 1 and 2, respectively. At the end of the study, the mean tumorgrowth inhibition in the SBT-100 treated group was 26.6% (p=0.29). FIG.8 illustrates the mean tumor volume. The tumor doubling times were 14.57days versus 18.19 days for Group 1 and Group 2, respectively. The %treatment/control for Group 2 was 74.8.

TABLE 30 Mean Body Weights for Mice in DU145 Phase Animal DosingRecovery Group ID 9/4 9/8 9/11 9/15 9/18 9/22 9/25 Control 001 29.6028.10 29.30 28.40 28.30 29.00 29.90 Control 002 29.70 30.10 31.20 30.1030.40 29.90 30.00 Control 003 30.80 30.10 31.00 31.70 31.20 31.10 31.10Control 004 26.30 25.70 26.60 25.20 26.10 26.20 25.80 Mean 29.10 28.5029.53 28.85 29.00 29.05 29.20 Median 29.65 29.10 30.15 29.25 29.35 29.4529.95 SD 1.94 2.09 2.13 2.78 2.29 2.09 2.33 % Change 0.00 −2.07 1.46−0.99 −0.37 −0.19 0.27 SBT-100 005 30.90 30.20 27.90 29.80 29.90 30.5030.10 SBT-100 006 28.40 26.20 27.30 26.90 27.50 29.10 28.50 SBT-100 00731.70 31.20 31.50 30.40 30.70 31.20 30.80 SBT-100 008 31.70 29.70 30.2028.20 28.10 28.80 29.00 Mean 30.68 29.33 29.23 28.83 29.05 29.90 29.60Median 31.30 29.95 29.05 29.00 29.00 29.80 29.55 SD 1.56 2.17 1.97 1.581.50 1.14 1.04 % Change 0.00 −4.47 −4.74 −6.00 −5.23 −2.39 −3.40

TABLE 31 Tumor Volumes for DU145 Phase Animal Pre-Dosing Dosing RecoveryGroup ID 8/24 8/27 8/31 9/3 9/4 9/8 9/11 9/15 9/18 9/22 9/25 Control 00139.18 41.27 38.41 92.80 93.22 121.16 203.79 310.41 409.15 430.31 450.89Control 002 45.05 35.99 64.98 95.50 103.83 135.42 225.62 327.76 478.14534.48 599.97 Control 003 46.65 22.37 88.76 127.98 141.49 213.24 384.15930.74 1,023.13 1,084.09 1,198.93 Control 004 17.06 36.44 65.73 131.20138.33 227.23 289.78 338.79 576.83 926.90 1,027.90 Mean 36.98 34.0264.47 111.87 119.22 174.26 275.84 476.93 621.81 743.95 819.42 Median42.11 36.22 65.36 111.74 121.08 174.33 257.70 333.27 527.49 730.69813.93 SD 13.67 8.12 20.58 20.53 24.32 53.70 80.91 302.77 276.25 311.67351.88 SBT- 005 33.80 23.32 35.67 86.02 89.21 151.92 145.67 386.92325.85 474.31 498.83 SBT- 006 59.44 41.00 54.21 98.56 121.39 148.44206.10 357.62 391.02 518.25 588.67 SBT- 007 42.30 35.11 77.90 144.06145.78 115.05 106.70 248.12 316.24 454.78 528.83 SBT- 008 69.37 50.1871.23 116.28 118.70 134.16 147.52 320.22 423.45 604.72 790.96 Mean 51.2337.40 59.75 111.23 118.77 137.39 151.50 328.22 364.14 513.01 601.83Median 50.87 38.06 62.72 107.42 120.05 141.30 146.60 338.92 358.43496.28 558.75 SD 16.13 11.25 18.90 25.16 23.17 16.76 40.98 59.97 51.6466.65 131.51 % T/C 0.00 −26.9 29.1 78.8 81.7 65.4 42.1 70.6 56.9 69.974.8 p-value 0.226 0.643 0.747 0.970 0.980 0.238 0.034 0.372 0.116 0.1970.291

In the PANC-1 tumor xenograft model, animals were randomized on day 22post-inoculation with a mean (±SE) tumor size of: 78.74±40.21 and93.84±36.31 for Groups 1 and 2, respectively. Mean body weights (±SE) atrandomization were: 22.50±1.47 and 24.23±1.63 for Groups 1 and 2,respectively. Table 32 summarizes the mean body weights (±SE) for entirestudy. At last day of dosing (Day 14), the mean tumor size (±SE) forcontrol group was 204.95±178.90 versus 159.03±28.01 for SBT-100 treatedmice. Table 33 summarizes the tumor volumes (±SE) for entire study. Atthe time of termination (day 28) mean tumor size (±S.E.) was:284.77±288.88 and 203.02±30.34, for groups 1 and 2, respectively. Meanbody weights (±SE) at termination were: 27.38±1.07 and 26.23±1.19 forGroups 1 and 2, respectively. At the end of the study, the mean tumorgrowth inhibition in the SBT-100 treated group was 41.78% (p=0.35). FIG.9 illustrates the mean tumor volume. The tumor doubling times were 18.51days versus 35.70 days for Group 1 and Group 2, respectively. The %treatment/control for Group 2 was 52.79.

TABLE 32 Mean Body Weights for Mice in PANC-1 Phase Animal DosingRecovery Group ID 9/9 9/11 9/15 9/18 9/22 9/25 9/29 Control 001 26.5026.60 25.80 27.10 25.70 26.10 27.20 Control 002 24.30 24.60 23.90 25.1024.40 25.00 25.60 Control 003 27.60 26.50 26.30 26.20 26.10 27.50 28.20Control 004 25.10 25.30 24.20 25.10 24.70 25.90 26.90 Mean 22.50 22.8023.04 24.30 24.58 25.90 27.38 Median 25.80 25.90 25.00 25.65 25.20 26.0027.05 SD 1.47 0.97 1.18 0.97 0.81 1.03 1.07 % Change 0.00 −0.39 −3.150.12 −2.41 1.05 4.33 SBT-100 005 22.60 22.80 21.40 22.50 22.60 22.9024.80 SBT-100 006 26.00 25.10 24.90 25.70 25.10 25.40 27.10 SBT-100 00723.10 22.30 22.40 22.70 23.10 23.50 25.70 SBT-100 008 25.20 25.00 25.2025.40 26.20 25.40 27.30 Mean 24.23 23.80 23.48 24.08 24.25 24.30 26.23Median 24.15 23.90 23.65 24.05 24.10 24.45 26.40 SD 1.63 1.46 1.87 1.711.69 1.29 1.19 % Change 0.00 −1.71 −3.14 −0.63 0.13 0.39 8.39

TABLE 33 Tumor Volumes for PANC-1 Phase Animal Pre-Dosing DosingRecovery Group ID 8/31 9/3 9/8 9/9 9/11 9/15 9/18 9/22 9/25 9/29 Control001 54.91 56.79 94.23 94.37 94.69 123.90 135.77 206.74 220.31 223.91Control 002 46.38 75.43 81.99 81.62 88.44 130.01 151.06 140.52 145.62202.22 Control 003 0.00 27.50 57.30 59.60 99.77 107.02 142.23 140.55168.68 187.27 Control 004 0.00 0.00 152.17 159.98 227.02 380.54 502.06514.93 574.44 781.45 Mean 20.26 32.54 78.74 80.91 104.18 151.29 189.83204.95 226.81 284.77 Median 23.19 42.15 88.11 87.99 97.23 126.95 146.64173.65 194.50 213.06 SD 29.45 33.13 40.21 43.18 66.52 130.48 179.63178.90 200.56 288.88 SBT- 005 39.60 64.75 76.44 78.07 57.54 93.17 112.98140.09 173.92 245.84 SBT- 006 40.31 37.27 68.57 73.13 76.46 113.30130.49 192.56 205.55 189.42 SBT- 007 85.71 91.27 147.61 149.02 123.95116.01 157.50 171.29 175.68 200.97 SBT- 008 48.72 55.19 82.73 83.1886.90 102.48 105.65 132.19 136.93 175.84 Mean 53.58 62.12 93.84 95.8586.21 106.24 126.65 159.03 173.02 203.02 Median 44.51 59.97 79.59 80.6281.68 107.89 121.73 155.69 174.80 195.19 SD 21.82 22.52 36.31 35.6927.94 10.49 23.05 28.01 28.10 30.34 % T/C 0.0 0.0 42.6 44.2 27.4 34.438.0 49.7 51.0 52.8 p-value 0.174 0.310 0.927 0.917 0.296 0.272 0.2860.350 0.343 0.355

Example 12: Efficacy of Anti-STAT3 Bacterial VHH13 (SEQ ID NO:3) sdAb inthe ER+/PR+(MCF-7) Human Breast Tumor Xenograft Model

This Example demonstrates the efficacy of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the MCF-7 human breast tumor xenograft model innude mice.

Female athymic nude mice (Crl:NU(Ncr)-Foxn1^(nu), Charles River) weretwelve weeks old with a body weight (BW) range of 23.0 to 30.1 g on Day1 of the study. The animals were fed and housed as described above.

MCF-7 human breast carcinoma cells were obtained and cultured asdescribed above, and used for the mouse xenograph. Three days prior totumor cell implantation, estrogen pellets (0.36 mg estradiol, 60-dayrelease, Innovative Research of America, Sarasota, Fla.) were implantedsubcutaneously between the scapulae of each test animal using asterilized trocar.

The tumor cells used for implantation were harvested during log phasegrowth and resuspended in phosphate buffered saline (PBS) at aconcentration of 1×10⁸ cells/mL. On the day of implantation, each testmouse received 1×10⁷ MCF-7 cells (0.1 mL cell suspension) implantedsubcutaneously in the right flank and tumor growth was monitored as theaverage size approached the target range of 100-150 mm³. Twenty-one dayslater, designated as Day 1 of the study, the animals were sorted intotwo groups each consisting of four mice with individual tumor volumesranging from 108 to 144 mm³ and group mean tumor volumes from 117 to 123mm.

Anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb was provided as apre-formulated ready to dose solution at a concentration 0.41867 mg/mLin 1 mL aliquots and were stored at −20° C. until needed. The 0.41867mg/mL solution provided 1 mg/kg dosage in a dosing volume of 23.88mL/kg. On each day of treatment, only needed vials of anti-STAT3bacterial VHH13 (SEQ ID NO:3) sdAb were thawed to room temperature. Allleftover dosing suspensions were retained at 4° C. as needed for thenext dose.

Two groups of athymic nude mice were dosed according to the protocolshown in Table 34. All vehicle (control) and anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb doses were administered intraperitoneally (i.p.)three times daily, six hours apart for fourteen days, with two dosesdelivered on Day 1 and one dose delivered on the morning of Day 15(tid×14, first day 2 doses). The dosing volume for vehicle andanti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb was 0.478 mL per 20 gramsof body weight (23.88 mL/kg) and was scaled to the body weight of eachindividual animal. Group 1 received the vehicle and served as thebenchmark group for tumor engraftment and progression, as well as thecontrol. Group 2 was given anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAbat 1 mg/kg.

TABLE 34 Protocol Design for the Study Treatment Regimen Group n Agentmg/kg Route Schedule 1 4 vehicle — ip tid × 14 first Day 2 doses 2 4VHH13 1 ip tid × 14 first Day 2 doses

Tumors were measured twice weekly, and each animal was euthanized whenits neoplasm reached the predetermined endpoint volume (1000 mm³) or atthe end of the study, day 39, whichever came first. When a tumor reachedthe endpoint volume, the animal was documented as euthanized for tumorprogression (TP), with the date of euthanasia. The time to endpoint(TTE) for each mouse was calculated by the following equation:

${TTE} = \frac{{\log_{10}\left( {{endpoint}\mspace{14mu} {volume}} \right)} - b}{m}$

where TTE is expressed in days, endpoint volume is expressed in mm³, bis the intercept, and m is the slope of the line obtained by linearregression of a log-transformed tumor growth data set. The data setconsists of the first observation that exceeded the endpoint volume usedin analysis and the three consecutive observations that immediatelypreceded the attainment of this endpoint volume. The calculated TTE isusually less than the TP date, the day on which the animal waseuthanized for tumor size. Animals that did not reach the endpointvolume were assigned a TTE value equal to the last day of the study(D39). Any animal classified as having died from treatment-related (TR)causes was to be assigned a TTE value equal to the day of death. Anyanimal classified as having died from non-treatment-related (NTR) causeswas to be excluded from TTE calculations.

Treatment efficacy was determined from tumor growth delay (TGD), whichis defined as the increase in the median TTE, in days, for a treatmentgroup compared to the control group:

TGD=T−C

The percent increase in the median TTE, relative to the control group,is

${\% \mspace{14mu} {TGD}} = {\frac{T - C}{C} \times 100}$

where:

T=median TTE for a treatment group, and

C=median TTE for the designated control group.

Treatment efficacy in each group may be indicated by the median tumorvolume, MTV(n), which was defined as the median tumor volume on the lastday of the study (D39) in the number of animals remaining (n) whosetumors had not attained the endpoint volume.

Treatment efficacy may also be determined from the incidence andmagnitude of regression responses observed during the study. Treatmentmay cause partial regression (PR) or complete regression (CR) of thetumor in an animal. In a PR response, the tumor volume was 50% or lessof its D1 volume for three consecutive measurements during the course ofthe study, and equal to or greater than 13.5 mm³ for one or more ofthese three measurements. In a CR response, the tumor volume was lessthan 13.5 mm³ for three consecutive measurements during the course ofthe study. Any animal with a CR response at the end of the study wasadditionally classified as a tumor-free survivor (TFS).

Animals were weighed daily for the first five days, then twice weeklyfor the remainder of the study. The mice were observed frequently forhealth and overt signs of any adverse treatment related TR side effects,and noteworthy clinical observations were recorded. Individual bodyweight loss was monitored per protocol, and any animal with weight lossexceeding 30% for one measurement, or exceeding 25% for threemeasurements, was to be euthanized for health as a TR death. If groupmean body weight recovered, dosing may resume in that group, but at alower dose or less frequent dosing schedule. Acceptable toxicity wasdefined as a group mean BW loss of less than 20% during the study andnot more than one TR death among ten treated animals, or 10%. Any dosingregimen resulting in greater toxicity is considered above the maximumtolerated dose (MTD). A death was to be classified as TR if it wasattributable to treatment side effects as evidenced by clinical signsand/or necropsy, or may also be classified as TR if due to unknowncauses during the dosing period or within 14 days of the last dose. Adeath was classified as NTR if there was evidence that the death wasrelated to the tumor model, rather than treatment-related. NTR deathsare further categorized as NTRa (due to accident or human error), NTRm(due to necropsy-confirmed tumor dissemination by invasion ormetastasis), and NTRu (due to unknown causes).

Prism 6.07 (GraphPad) for Windows was employed for graphical analyses.Statistics were not employed due to small sample size.

A scatter plot was constructed to show TTE values for individual mice,by group; this plot shows NTR deaths, which were excluded from all otherfigures. Individual animal, group median and mean tumor volumes wereplotted as functions of time. When an animal exited the study because oftumor size or TR death, its final recorded tumor volume was includedwith the data used to calculate the median volume at subsequent timepoints. A Kaplan-Meier plot was constructed to show the percentage ofanimals in each group remaining on study versus time. Tumor growthcurves were truncated after two TR deaths occurred in the same group.Group mean BW changes over the course of the study were graphed aspercent change, ±SEM, from Day 1. Tumor growth and BW change curves weretruncated after more than half the assessable mice in a group exited thestudy. FIG. 10 illustrates the mean tumor volume in the study.

Table 35 provides the mean BW losses, TR and NTR deaths for the mice.Clinical signs were recorded when observed, as shown in Tables 36-38. NoTR deaths occurred during the study. Bodyweight losses were variable,severe for one animal in each group, and resulted from estrogen effects.Clinical observations including weight loss, enlarged uterine horns, andbladder crystals were present in both groups and were also attributableto estrogen effects. Estrogen toxicity resulted in two non-treatmentrelated deaths in each group. The treatment evaluated in the study wasacceptably tolerated.

TABLE 35 Response Summary Treatment Regimen Median % MTV(n) RegressionsMean BW Deaths Group n Agent mg/kg Route Schedule TTE T-C TGD D 39 PR CRTFS Nadir TR NTR 1 2 Vehicle — ip tid x 14 first 23.2 — — — 0 0 0 −15.6%0 2 Day 2 doses Day 25 2 2 VHH13 1 ip tid x 14 first 32.9 9.7 42 — 0 0 0−21.8% 0 2 Day 2 doses Day 32

TABLE 36 Body Weight Body Weight Date Jul. Jul. Jul. Jul. Jul. Aug. Aug.Aug. Aug. Aug. Aug. Aug. Aug. Aug. Sep. 27, 28, 29, 30, 31, 3, 6, 10,13, 17, 20, 24, 27, 31, 3, 2015 2015 2015 2015 2015 2015 2015 2015 20152015 2015 2015 2015 2015 2015 Day of Study 1 2 3 4 5 8 11 15 18 22 25 2932 36 39 Wt Wt Wt Wt Wt Wt Wt Wt Wt Wt Wt Wt Wt Wt Wt A# (g) (g) (g) (g)(g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) Group I: vehicle (ip, tid x14 first Day 2 doses) 1 27.50 28.60 28.10 29.30 29.40 NTRa on Aug. 1,2015 2 26.30 27.30 27.40 27.30 27.40 26.80 26.40 26.50 26.60 27.20 TP onAug. 17, 2015 3 30.10 31.00 30.50 30.00 31.10 29.50 28.20 26.00 22.5023.60 23.80 TP on Aug. 20, 2015 4 23.00 24.20 24.40 25.00 NTRu on Aug.3, 2015 Mean 26.7 27.8 27.6 27.9 28.2 28.2 27.3 26.3 24.6 25.4 23.8STDEV 2.9 2.8 2.5 2.2 2.6 1.9 1.3 0.4 2.9 2.5 n 4 4 4 4 4 2 2 2 2 2 1Group 2: VHH13 (1 mg/kg, ip, tid x 14 first Day 2 doses) 1 28.90 29.3028.50 29.60 28.80 28.70 27.80 28.30 28.20 28.80 29.00 28.90 TP on Aug.24, 2015 2 25.30 27.00 26.40 26.40 26.30 25.90 25.80 26.00 24.50 21.9020.10 21.10 21.20 23.80 24.20 3 27.20 25.40 23.90 NTRu on Jul. 30, 20154 27.60 27.50 27.10 27.30 27.20 26.90 26.10 NTRu on Aug. 8, 2015 Mean27.3 27.3 26.5 27.8 27.4 27.2 26.6 27.2 26.4 25.4 24.6 25 21.2 23.8 24.2STDEV 1.5 1.6 1.9 1.7 1.3 1.4 1.1 1.6 2.6 4.9 6.3 5.5 n 4 4 4 3 3 3 3 22 2 2 2 1 1 1

TABLE 37 Tumor Measurement Caliper Measurement Date Jul. 27, Jul. 30,Aug. 3, Aug. 6, Aug. 10, Aug. 13, Aug. 17, 2015 2015 2015 2015 2015 20152015 Day of Study 1 4 8 11 15 18 22 W L W L W L W L W L W L W L A# (mm)(mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) Group1: vehicle (ip, tid x 14 first Day 2 doses) 1 6 6 7 8 NTRa on Aug. 1,2015 2 5 9 7 9 8 12 9 13 10 13 10  13 12 15 3 6 7 7 10 9 10 10  12 11 1211  12 11 13 4 6 8 7 11 NTRa on Aug. 3, 2015 Group 2: VHH13 (1 mg/kg,ip, tid x 14 first Day 2 doses) 1 6 6 7 8 8 10 9 10  9 10 9 10 10 11 2 66 6 7 7  8 7  8  8  9 8  9  9 10 3 6 7 NTRu on Jul. 30, 2015 4 6 7 6 8 710 8 10 Date Aug. 20, Aug. 24, Aug. 27, Aug. 31, Sep. 3, 2015 2015 20152015 2015 Day of Study 25 29 32 36 39 W L W L W L W L W L A# (mm) (mm)(mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) Group 1: vehicle (ip, tid x 14first Day 2 doses) 1 NTRa on Aug. 1, 2015 2 TP on Aug. 17, 2015 3 12 14TP on Aug. 20, 2015 4 NTRa on Aug. 3, 2015 Group 2: VHH13 (1 mg/kg, ip,tid x 14 first Day 2 doses) 1 12 12 13 13 TP on Aug. 24, 2015 2  9 10 1010 10 10 12 12 13 13 3 4 NTRu on Aug. 8, 2015

TABLE 38 Tumor Volume Tumor Volume Date Jul. Jul. Aug. Aug. Aug. Aug.Aug. Aug. Aug. Aug. Aug. Sep. 27, 30, 3, 6, 10, 13, 17, 20, 24, 27, 31,3, 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 Day ofStudy 14 8 11 15 18 22 25 29 32 36 39 TV TV TV TV TV TV TV TV TV TV TVTV A# (mm³) (mm³) (mm³) (mm³) (mm³) (mm³) (mm³) (mm³) (mm³) (mm³) (mm³)(mm³) Group 1: vehicle (ip, tid x 14 first Day 2 doses) 1 108 196 NTRaon Aug. 1, 2015 2 113 221 384 527 650 650 1080 TP on Aug. 17, 2015 3 126245 405 600 726 726 787 1008 TP on Aug. 20, 2015 4 270 NTRu on Aug. 3,2015 Mean 122.6 232.8 394.5 563.3 688 688 933.3 1008 SEM 8.1 15.8 10.536.8 38 38 146.8 n 4 4 222221 Group 2: VHH13 (1 mg/kg, ip, tid x 14first Day 2 doses) 1 108 196 320 405 405 405 550 864 1099 TP on Aug. 24,2015 2 108 126 196 196 288 288 405 405 500 500 864 1099 3 126 NTRu onJul. 30, 2015 4 126 144 245 320 NTRu on Aug. 8, 2015 Mean 117 155.3253.7 307 346.5 346.5 477.5 634.5 799.3 500 864 1098.5 SEM 5.2 21 36.160.7 58.5 58.5 72.5 229.5 299.3 n 4 3 3 322222111

Table A1 MCF-7-e353 Clinical Observations

Because two out of the four mice in the control group and also in thetreatment group died of estrogen toxicity, no statistical conclusioncould be determined. With the data available, the median tumor growthand mean tumor volume were reduced in the treatment group when comparedto the control group. This difference was present during the 14 days oftreatment but also to day 25 of the study. It took the control group 25days to reach a tumor volume of 1000 mm³, whereas the treatment grouptook 36 days to reach a tumor volume of 1000 mm³. This suggests thatanti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb slows the growth of MCF-7tumor in vivo. Throughout the study both the control group and thetreatment group maintained similar weights. This suggests that theanti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb did not cause toxicitywith respect to weight loss.

Example 13: Treatment of Human HER2+(BT474) Breast Cancer withAnti-STAT3 Bacterial VHH13 (SEQ ID NO:3) sdAb in Xenograft Mice

In this Example, the efficacy of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb was determined in the BT474 human breast tumor xenograft inCB.17 SCID mice.

Two groups of 8-12 week old CB.17 SCID mice containing xenographs of 1mm³ BT474 tumor fragments in their flank were treated according to theprotocol shown in Table 39 when the tumors reached an average size of100-150 mm³. All vehicle (PBS control) and anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb (shown in Table 39 as SB-01) doses were administeredintraperitoneally (i.p.) three times daily, six hours apart for fourteendays, with two doses delivered on Day 1 (tid×14, first day 2 doses). Thedosing volume for vehicle and anti-STAT3 bacterial VHH13 (SEQ ID NO:3)sdAb was 0.478 mL per 20 grams of body weight (23.88 mL/kg) and wasscaled to the body weight of each individual animal. Group 1 receivedthe vehicle and served as the benchmark group for tumor engraftment andprogression, as well as the control. Group 2 was given anti-STAT3bacterial VHH13 (SEQ ID NO:3) sdAb at 1 mg/kg.

TABLE 39 Study protocol Regimen 1 Group N Agent Vehicle mg/kg RouteSchedule  1# 4 vehicle — ip tid x 14 first day 2 doses 2 4 SB-01 1 iptid x 14 first day 2 doses

During the first 14 days of the study, the treatment group receivedanti-STAT3 B VHH13 and the control group only received the vehicle. Asshown in Table 40, during this time, the treatment group maintained andgained weight throughout the study while the control group had lowerweights throughout the study. This suggests that the treatment group didnot experience toxicity from anti-STAT3 bacterial VHH13 (SEQ ID NO:3)sdAb with respect to weight loss. Both groups mean tumor volume andmedian tumor volume were similar, and exactly the same on day 15 of thestudy. On day 59 of the study, both groups reached a tumor volume of 700cubic mm³. This suggests that the anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb did not reduce the growth of BT474 tumors in vivo whencompared to the control group. FIG. 11 illustrates the group mean tumorvolume.

TABLE 40 BT474 Response Summary Treatment Regimen 1 MTV Vehi- mg/ Median% Stat (n) BW NTR Group n Agent cle kg Route Schedule TTE T-C TGD SignDay 60 PR CR TFS Nadir TR NTRm NTR  1# 4 vehicle — ip tid x 14 49.2 — —288 (2) 0 0 0 −9.1% (3) 0 0 0 first day 2 doses 2 4 SB-01 1 ip tid x 1460.0 10.8 22 550 (3) 0 0 0 — 0 0 0 first day 2 doses #Control Group

Example 14: Production of Mouse Monoclonal Antibody Directed AgainstAnti-STAT3 Bacterial VHH13 (SEQ ID NO:3) sdAb

In this Example, mouse monoclonal antibodies were generated towards thesdAb of the invention. The animals used were BALB/c female mice, 8-10week. A water-soluble adjuvant was used (CBL). The HAT and the HT usedwere from Sigma-Aldrich.

Anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb was used to immunize threemice and make hybridoma cell lines. The mice were immunized three timeseach with water-soluble adjuvant. In one mouse, the serum titer reached1/51200. The mouse was sacrificed and hybridoma cell lines were made byfusing spleen cells with myeloma cell line Sp2/0.

The fused cells were seeded into 96 well plates by limited dilution. Thefused cells were cultured in the presence of HAT, and 651 single cloneswere tested. Of the 651 single clones, 27 positive clones wereidentified that specifically bound to anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb antigen.

Example 15: Cytotoxicity of KRAS (G12D) Single Domain Antibodies onPANC-1 Human Pancreatic Cancer Cells

This Example demonstrates the anti-proliferative effects of theanti-KRAS (G12D) (SEQ ID NO:2) sdAb using the human pancreatic cancercell line PANC-1. For the experiments, the PANC-1 cells were grown untilthey reached a confluency of 90%. At that time, proliferation studieswere carried out using the MTT assay as described above.

The anti-proliferative properties of anti-KRAS (G12D) (SEQ ID NO:2) sdAbon PANC-1 cells three days post treatment are shown in Table 41. PANC-1cells treated with the anti-KRAS (G12D) (SEQ ID NO:2) sdAb showed anaverage growth inhibition of 19.9 and 37.7 at 50.0 and 100 μg/ml,respectively.

TABLE 41 Anti-proliferative Actions of Anti-KRAS (G12D) (SEQ ID NO: 2)sdAb on PANC-1 Cancer Cells Mean Abs ± SE % Inhibition control 0.281 ±0.017  50 μg/ml 0.225 ± 0.006 19.9 100 μg/ml 0.175 ± 0.016 37.7

Thus, the anti-KRAS (G12D) (SEQ ID NO:2) sdAb showed dose-dependentgrowth inhibition in the PANC-1 human pancreatic cancer cells.

Example 16: In Vitro Growth Inhibition by TNF-Alpha sdAb

This Example demonstrates the method development to determine TNF-alphaconcentration and evaluation of the inhibition of TNF-alpha function.The concentration of TNF-alpha required to show measurable modulation ofactivity in the U937 human lung lymphoblast cell line was evaluated byquantitation of the ATP present, which signals the presence ofmetabolically active cells using Promega's Cell Titer-GJo® LuminescentCell Viability assay.

The U937 cells were seeded in a clear polystyrene 96-well microcultureplate (Corning® Costar® 96-well flat bottom plate, Cat. #3997) in atotal volume of 90 μL/well. After 24 hours of incubation in a humidifiedincubator at 37° C. with 5% CO₂ and 95% air, 5 μL of 20×, seriallydiluted TNF-alpha in growth medium was added to each well in duplicate(10 pt dose response, highest concentration 20 ng/mL). Additionally, 5μL of 20×, diluted staurosporine in growth medium was added to each wellin duplicate (concentration 1 nM).

After 24 hours of culture in the presence of test agents, theconcentration of compound required to show measurable modulation ofTNF-alpha activity in the U937 cell line as evaluated by quantitation ofthe ATP present. Percent cell growth was calculated relative tountreated control wells. All tests were performed in duplicate at eachconcentration level.

The EC₅₀ value for the test agents was estimated using Prism 6.05 bycurve-fitting the data using the following four parameter-logisticequation:

$Y = {\frac{{Top} - {Bottom}}{1 + \left( {{X/1}\; C_{50}} \right)^{n}} + {Bottom}}$

where Top is the maximal % of control absorbance, Bottom is the minimal% of control absorbance at the highest agent concentration, Y is the %of control absorbance, X is the agent concentration, IC₅₀ is theconcentration of agent that inhibits cell growth by 50% compared to thecontrol cells, and n is the slope of the curve.

FIGS. 12 and 13 demonstrate that TNF-alpha is cytotoxic to the U937cells. The IC₅₀ for TNF-alpha against U937 is 95.10 pg/ml. The TNF-alphacurve shows a dose titration killing effect.

FIG. 14 demonstrates that TNF-alpha cytotoxicity against U937 isinhibited by the three different anti-TNF-alpha VHHs. Whenanti-TNF-alpha VHH62 (SEQ ID NO:47) sdAb, anti-TNF-alpha VHH 66 (SEQ IDNO:45) sdAb, and anti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb were incubatedwith a constant dose of TNF-alpha, at EC₅₀, all three anti-TNF-alphaVHHs inhibit killing of U937 by TNF-alpha. The IC₅₀ of anti-TNF-alphaVHH62 (SEQ ID NO:47) sdAb was approximately 713.6 ug/ml. The IC₅₀ ofanti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb was greater than 208.055 ug/ml.The IC₅₀ of anti-TNF-alpha VHH66 (SEQ ID NO:45) sdAb could not bedetermined because it completely inhibited the cytotoxicity of TNF-alphafrom concentrations of about 1×10² ug/ml to 1×10² ug/ml ofanti-TNF-alpha VHH66 (SEQ ID NO:45) sdAb. In this concentration range ofanti-TNF-alpha VHH66 (SEQ ID NO:45) sdAb, there is an increase in U937cell growth, and thus complete inhibition of TNF-alpha activity.

Example 17: Anti-Proliferative Effects of STAT3 VHH13 (SEQ ID NO:3) sdAbon Glioblastoma, Osteosarcoma and Fibrosarcoma Cell Lines

The anti-proliferative effects of the STAT3 VHH13 (SEQ ID NO:3) sdAb wasassayed using HT1080, SJSA and U87 mg cells. The treatment time and doseis shown in Table 42, as is the percent growth inhibition is shown inTable 42. The IC₅₀ of STAT3 VHH13 (SEQ ID NO:3) sdAb in HT1080 cells was33 μg/ml. The IC₅₀ STAT3 VHH13 (SEQ ID NO:3) sdAb for SJSA cells was 51μg/ml. The IC₅₀ of anti-STAT3 VHH13 (SEQ ID NO:3) sdAb for U87 mg cellswas 65 μg/ml. The results show statistically significant suppressionwith anti-STAT3 VHH13 (SEQ ID NO:3) sdAb within three days aftertreatment.

TABLE 42 Growth Inhibition Dose (μg/ml) Absorbance ± S.E. % InhibitionHT1080 cells treated for 72 hr. with SBT-100 0 1.58 ± 0.07 12.5 1.36 ±0.09 14 25 1.09 ± 0.01 31 50 0.42 ± 0.04 73 100 0.27 ± 0.01 83 200 0.22± 0.02 86 SJSA cells treated for 72 hr. with SBT-100 0 1.52 ± 0.15 12.51.52 ± 0.10 0 25 1.36 ± 0.09 11 50 0.90 ± 0.04 41 100 0.27 ± 0.01 82 2000.26 ± 0.01 83 U87mg cells treated for 72 hr. with SBT-100 0 0.555 ±0.02 12.5 0.702 ± 0.04 0 25 0.687 ± 0.03 0 50 0.456 ± 0.01 18 100 0.271± 0.02 51 200 0.211 ± 0.03 62

Example 18: Anti-Proliferative Effects of STAT3 VHH13 (SEQ ID NO:3)sdAbs on Human Cancer Cell Lines in Combination with Chemotherapy

The anti-proliferative effects of the STAT3 VHH13 (SEQ ID NO:3) sdAb wasassayed using the human cancer cell line PANC-1 with or without thechemotherapy drug Gemcitabine.

The PANC-1 cell line was maintained as described above. The cells weretreated with STAT3 VHH13 (SEQ ID NO:3) sdAb (denoted SBT-100) alone,Gemcitabine alone, or with a combination of STAT3 VHH13 (SEQ ID NO:3)sdAb and Gemcitabine. Addition of 10 micromolar Gemcitabine (IC₅₀concentration) showed a 48% growth inhibition of PANC-1 cells three daysafter treatment. When 100 μg/ml STAT3 VHH13 (SEQ ID NO:3) sdAb was addedto the cells, there was a 68% growth inhibition of PANC-1 cells threedays after treatment. The combination of 10 micromolar Gemcitabine and100 μg/ml STAT3 VHH13 (SEQ ID NO:3) sdAb resulted in an 80% growthinhibition of PANC-1 cells three days after treatment. Similarly, thecombination of Gemcitabine (at ⅛th it's IC50 conc.) and 100 μg/ml STAT3VHH13 (SEQ ID NO:3) sdAb resulted in a 75% growth inhibition of PANC-1cells three days after treatment.

Table 43 shows the growth inhibition of PANC-1 cells treated for 72hours with STAT3 VHH13 (SEQ ID NO:3) sdAb (100 μg/ml) alone or incombination with Gemcitabine (5 or 10 μM) prepared in water.

TABLE 43 Growth Inhibition of PANC-1 Cells Treatment % Inhibitionp-value Vehicle SBT-100 77 p < 0.001 Gemcitabine (5 μM) 38 p < 0.001Gemcitabine (10 μM) 35 p < 0.001 SBT-100 + Gem (5 μM) 87 p < 0.001SBT-100 + Gem (10 μM) 81 p < 0.001

The growth inhibition was dose dependent, as shown in Table 44. ofPANC-1 cells treated for 72 hours with STAT3 VHH13 (SEQ ID NO:3) sdAbalone or in combination with Gemcitabine prepared in DMSO.

TABLE 44 Growth Inhibition of PANC-1 Cells Treatment Absorbance ±SE %Inhibition P-value Vehicle Control 1.050 0.075 Gemcitabine 10 μM 0.5430.037 48 p < 0.001 Gemcitabine 5 μM 0.631 0.012 40 p < 0.001 Gemcitabine2.5 μM 0.681 0.017 35 p < 0.01 Gemcitabine 1.25 μM 0.861 0.077 18 nsGemcitabine 0.625 μM 1.050 0.162 0 ns Gemcitabine 0.313 μM 0.997 0.054 5ns Gemcitabine 0.156 μM 1.210 0.062 0 ns Gemcitabine 0.078 μM 1.2300.039 0 ns Gemcitabine 0.039 μM 1.240 0.014 0 ns Gemcitabine 0.020 μM1.560 0.101 0 ns SBT-100 100 μg/ml 0.333 0.050 68 p < 0.001 Gemcitabine10 μM + SBT-100 100 μg/ml 0.234 0.041 78 p < 0.001 Gemcitabine 5 μM +SBT-100 100 μg/ml 0.294 0.044 72 p < 0.001 Gemcitabine 2.5 μM + SBT-100100 μg/ml 0.240 0.036 77 p < 0.001 Gemcitabine 1.25 μM + SBT-100 100μg/ml 0.204 0.013 81 p < 0.001 Gemcitabine 0.625 μM + SBT-100 100 μg/ml0.232 0.008 78 p < 0.001 Gemcitabine 0.313 μM + SBT-100 100 μg/ml 0.2760.010 74 p < 0.001 Gemcitabine 0.156 μM + SBT-100 100 μg/ml 0.290 0.04772 p < 0.001 Gemcitabine 0.078 μM + SBT-100 100 μg/ml 0.265 0.015 75 p <0.001 Gemcitabine 0.039 μM + SBT-100 100 μg/ml 0.446 0.039 56 p < 0.001Gemcitabine 0.020 μM + SBT-100 100 μg/ml 0.457 0.002 56 p < 0.001

Example 19: Treatment of PANC-1 Cells with Anti-STAT3 Bacterial VHH13(SEQ ID NO:3) sdAb in Xenograft Mice

In this Example, the efficacy of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb was determined in PANC-1 xenograft in mice. The 1.5×10⁶PANC-1 cells were injected into nude mice, and large xenograft tumorswere grown to between 100-150 mm³. The mice were divided into fourgroups. As shown in Table 45, the groups were left untreated, treatedfor 14 days with STAT3 VHH13 (SEQ ID NO:3) sdAb alone, treated withGemcitabine alone, or treated with a combination of STAT3 VHH13 (SEQ IDNO:3) (SBT-100) sdAb and Gemcitabine, followed by a 7 day recoveryperiod. Body weight (Table 46) and inhibition of tumor growth (Table 47)was measured on days 1, 5, 8, 12, 15, 19, and 22.

TABLE 45 Treatment # of cells Mean Tumour inoculated/ # of Dose Size(mm³) Model mouse Group mice Agent (mg/kg) Route Schedule Day 1 PANC-11.5 × 10⁶ 1 10 Control (PBS) 0 IP BIDx14 125.31 ± 16.5  2 6 STB-100 5 IPBIDx14 107.34 ± 29.46 3 6 Gemcitabine 20 IP QDx7 122.23 ± 23.46 4 6SBT-100 + 5 + 20 IP BIDx14 + 115.87 ± 10.01 Gemcitabine QDx7

As shown in Table 46, there was no substantial weight loss observed inmice after the different treatment regimens.

TABLE 46 Body Weight BODY Day 1 Day 5 Day 8 Day 12 Day 15 Day 19 Day 22WEIGHT (g) Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM MeanSEM Control 23.84 0.81 24.41 0.89 24.02 0.80 24.06 0.91 23.73 0.86 24.220.92 24.55 0.87 5 mg/g 23.68 0.50 24.35 0.72 23.72 0.62 23.25 0.47 23.720.48 24.38 0.50 24.63 0.43 SBT-100 BID 20 mg/kg 23.77 0.68 23.47 0.6023.37 0.46 23.13 0.43 212.98 0.56 23.97 0.37 24.55 0.46 Gemcitabine 5mg/kg 22.93 0.90 22.30 0.80 22.03 0.76 21.68 0.63 21.85 0.80 22.85 0.8223.53 0.81 SBT-100 + 20 mg/kg Gemcitabine

PANC-1 tumors demonstrated enhanced inhibition after treatment witheither STAT3 VHH13 (SEQ ID NO:3) sdAb alone or Gemcitabine alone.However, treatment with a combination of STAT3 VHH13 (SEQ ID NO:3) sdAband Gemcitabine resulted in a synergistic inhibition of tumor growth, asseen in Table 48. Combination treatment resulted in increased inhibitionof tumor growth as compared to the inhibition seen with either compoundalone.

TABLE 47 Tumor Growth TUMOR VOLUMES Day 1 Day 5 Day 8 Day 12 Day 15 Day19 Day 22 (mm³) Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEMMean SEM p Value Control 125.31 16.15 188.50 22.66 200.72 24.42 238.0521.52 320.80 34.54 407.55 42.23 483.19 51.64 5 mg/g 107.34 29.46 145.8628.86 149.12 43.88 188.78 31.64 233.25 56.43 332.68 82.01 390.59 115.03p < −0.05 SBT-100 BID 20 mg/kg 122.23 23.46 165.57 41.17 183.88 25.61191.6 35.30 246.48 27.01 312.74 40.96 411.93 43.33 p < −0.01 Gemcitabine5 mg/kg 115.87 10.01 152.90 19.60 169.92 23.99 169.42 14.14 207.00 19.43259.31 45.72 330.88 34.96 p < −0.001 SBT-100 + 20 mg/kg Gemcitabine

Table 48 depicts the percent inhibition of tumor growth after theindicated treatment. The combination of STAT3 VHH13 (SEQ ID NO:3) sdAband Gemcitabine resulted in an increased inhibition of tumor growth ascompared to cells treated with either STAT3 VHH13 (SEQ ID NO:3) sdAb orGemcitabine alone.

TABLE 48 Inhibition of Tumor Growth % INHIBITION OF CONTROL Day 12 Day15 Day 19 Day 22 5 mg/kg SBT-100 BID 20.70 27.29 18.37 19.17 20 mg/kgGemcitabine 19.51 23.17 23.26 14.93 5 mg/kg SBT-100 + 20 mg/kg 28.8335.48 36.37 31.52 Gemcitabine

Example 20: STAT3 VHH13 (SEQ ID NO:3) sdAb is Localized in Brain andTumor Cells

Various tissues from the xenograft mice with a MDA-MB-231 tumor asdescribed in Example 11 were taken for immunohistochemical analysis.Slides were made from the tissues using standard procedures. The slideswere then blocked with blocking serum. A 1:50 dilution of the primaryantibody, in this case a STAT3 rabbit monoclonal antibody (CellSignaling Inc., Danvers, Mass., Catalog #4904), was made with buffercontaining 1.5% blocking buffer for 1.5 hours at room temperature in ahumidity chamber. The slides were then rinsed with phosphate bufferedsaline (PBS). The slides were then incubated in prediluted biotinylatedpanspecific universal secondary antibody for 10 minutes, followed by aPBS rinse. The slides were then incubated in streptavidin/peroxidasecomplex reagent for 5 minutes, followed by a PBS rinse. The slides werethen incubated in peroxidase substrate solution for 15 minutes, followedby a rinse in distilled water. The slides were counterstained withGill's #1 hematoxylin for 45 seconds, and then rinsed three times withwater for five minutes followed by 45 seconds with Scott's tap watersubstitute.

As seen in FIG. 15, anti-STAT3 VHH13 (SEQ ID NO:3) sdAb can be seen inneurons and glial cells, indicating that anti-STAT3 VHH13 (SEQ ID NO:3)sdAb can cross the blood-brain barrier. Anti-STAT3 VHH13 (SEQ ID NO:3)sdAb can also be seen in the cytoplasm of the living cancer cells, ascan be seen in FIG. 16. These results show that anti-STAT3 VHH13 (SEQ IDNO:3) sdAb can cross the cell membrane into cells without exogenoustargeting sequences or compounds.

Example 21: Internalization of Anti-STAT3 VHH13 (SEQ ID NO:3) sdAb andTNF-Alpha VHH66 (SEQ ID NO:45) sdAb in MDA-MB 231 and PANC-1 Cells

MDA-MB 231 and PANC-1 cell lines were plated at low density on chamberslides and left overnight for cell attachment. The next day, thechambers were rinsed with fresh media and STAT3 VHH13 (SEQ ID NO:3) sdAb(diluted 1:10 in media) or TNF-alpha VHH66 (SEQ ID NO:45) sdAb was addedto the chamber. An anti-HIV-1 Reverse Transcriptase (RT) sdAb was usedas a negative control.

The slides were incubated for 18 hours after sdAb addition. The chamberswere briefly rinsed with media and fixed in ice-cold methanol for 5-10min. The chambers were then dried and stained with a fluorescentanti-His tag antibody (1:200)(Novus Biologicals, Littleton, Colo.) for45 min at room temperature. The slides were rinsed in PBS for 5 minutesand mounted.

Both the MDA-MB 231 cells and PANC-1 cells incubated with STAT3 VHH13(SEQ ID NO:3) sdAb showed positive cytoplasmic staining, as shown inFIGS. 17 and 18 respectively. Similarly, both the MDA-MB 231 cells andPANC-1 cells incubated with TNF-alpha VHH66 (SEQ ID NO:45) sdAb showedpositive cytoplasmic staining, as shown in FIGS. 19 and 20,respectively. As expected, no staining was seen in either cell line withthe anti-HIV-1 RT sdAb.

Example 22: Anti-STAT3 VHH13 (SEQ ID NO:3) sdAb Binds KRAS, Mutant KRASand Mutant STAT3

Protein binding experiments were performed on a Biacore 3000 (GeneralElectric Company, Fairfield, Conn.) at 25° C. The assay buffer contained10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.05% P20. Theregeneration buffer contained 10 mM glycine HCl pH 1.75, and theimmobilization buffer contained 10 mM sodium acetate, pH 5.0. The flowrate used for capturing the ligand was 5 ul/min. The flow rate used forkinetics analysis was 30 ul/min.

The ligands used for the protein binding experiment were human KRAS andhuman STAT3. The ligands were directly immobilized by amine coupling(EDC/NHS) at a response unit (RU) of 1000, 420, and 1800 on flow cell 2and 3 for KRAS and 4 for STAT3, respectively, on a CMS sensor chip. Flowcell 1 was kept blank and used for background subtraction. Theun-occupied sites on the CMS chip were blocked with 1M ethanol amine.For binding analysis, the analyte, anti-STAT3 VHH13 (SEQ ID NO:3) sdAbor anti-KRAS (G12D) (SEQ ID NO:2) sdAb was flowed over the sensor chip.Binding of analyte to the ligand was monitored in real time. Theaffinity constant (KD=kd/ka) was calculated from the observed on rate(ka) of off rate (kd), as shown in Table 49. Full kinetic analysis wasperformed at analyte concentrations as indicated. Chi square analysiswas carried out between the actual sensorgram and the sensorgramgenerated from the BIAnalysis software to determine the accuracy of theanalysis. A chi square value within 1-2 is considered accurate and below1 is highly accurate.

The anti-STAT3 VHH13 (SEQ ID NO:3) sdAb bound both KRAS and STAT3ligands. The anti-KRAS (G12D) (SEQ ID NO:2) sdAb anti-KRAS VHH analytebound the KRAS ligand, but not the STAT3 ligand.

TABLE 49 Kinetic Analysis Ligand Analyte ka (1/Ms) kd(1/s) Rmax KD (M)Conc. (nM) Chi2 Hu.KRAS Anti-STAT 3.66 × 10⁴ 4.62 × 10⁻³ 11.8 1.26 ×10⁻⁷ 0 0.0783 1000 RU VHH13 12.5 Flow cell 2 25 50 100 100 200 Hu.KRASAnti-KRAS 3.96 × 10³ 4.56 × 10⁻³ 358 1.15 × 10⁻⁶ 0 0.223 420 RU VHH 12.5Flow cell 3 25 50 100 100 200 Hu.STAT3 Anti-STAT 7.58 × 10⁴ 1.70 × 10⁻³17.6 2.24 × 10⁻⁸ 0 0.194 1800 RU VHH13 12.5 Flow cell 4 25 50 100 100200 Hu.STAT3 Anti-KRAS NA NA NA NA 0-200 NA 1800 RU VHH

Example 23: Anti-STAT3 VHH13 (SEQ ID NO:3) sdAb Binds KRAS, Mutant KRASand Mutant STAT3

Protein binding experiments were performed on a Biacore 3000 asdescribed above. The ligands used for the protein binding experimentwere anti-STAT5-31 sdAb (SEQ ID NO:83), anti-KRAS (G12D) (SEQ ID NO:2)sdAb and anti-STAT3 VHH13 (SEQ ID NO:3) sdAb. The ligands were directlyimmobilized by amine coupling (EDC/NHS) on a CMS sensor chip at aresponse unit (RU) of 440, 140, and 1000 on flow cells 2, 3 and 4,respectively. Flow cell 1 was kept blank and used for backgroundsubtraction. The un-occupied sites on the CMS chip were blocked with 1Methanol amine. For binding analysis, the analyte, STAT5, normal humanKRAS, or mutant human KRAS (12 ASP) was flowed over the sensor chip.Binding of analyte to the ligand was monitored in real time. Theaffinity constant (KD=kd/ka) was calculated from the observed on rate(ka) of off rate (kd). Full kinetic analysis was performed at analyteconcentrations as indicated. Chi square analysis was carried out betweenthe actual sensorgram and the sensorgram generated from the BIAnalysissoftware to determine the accuracy of the analysis. A chi square valuewithin 1-2 is considered accurate and below 1 is highly accurate.

As shown in Table 50, STAT3, normal and mutant KRAS, as well as STAT5ligands bound the anti-STAT3 VHH13 (SEQ ID NO:3) sdAb ligand, but notSTAT5-31 VHH. anti-KRAS (G12D) (SEQ ID NO:2) sdAb bound the human KRASprotein, but not STAT5 or mutant KRAS (12 ASP).

TABLE 50 Kinetic Analysis Ligand Analyte ka (1/Ms) kd(1/s) Rmax KD (M)Conc. (nM) Chi2 Result STAT5-31 VHH STAT5 NA NA NA NA 500 NA No binding(440 RU) protein STAT5-31VHH hu.KRAS NA NA NA NA 500 NA No binding (440RU) full STAT5-31 VHH hu.KRAS12 NA NA NA NA 500 NA No binding (440 RU)ASP anti-KRAS (G12D) STAT5 NA NA NA NA 500 NA No binding (SEQ ID NO: 2)protein sdAb (140 RU) anti-KRAS (G12D) hu.KRAS 1.86 × 10⁵ 5.52 × 10⁻³8.94 2.96 × 10⁻⁸ 500 0.42 Binding (SEQ ID NO: 2) full sdAb (140 RU)protein Anti-KRAS (G12D) Hu.KRAS NA NA NA NA 500 NA No binding VHH (140RU) 12ASP Anti-STAT3 STAT5 2.99 × 10⁴ 7.90 × 10⁻⁴ 27.2 2.64 × 10⁻⁸ 5000.0719 Binding VHH13 (1000 RU) protein Anti-STAT3 Hu.KRAS 5.09 × 10⁴2.76 × 10⁻⁴ 36 5.42 × 10⁻⁹ 500 0.338 Binding VHH13 (1000 RU) fullprotein Anti-STAT3 Hu.KRAS 2.62 × 10⁴ 8.06 × 10⁻⁷ 12.9  3.08 × 10⁻¹¹ 5000.0546 Binding VHH13 (1000 RU) 12ASP

Example 24: Activity of Anti-STAT3 sdAbs

To determine whether the anti-STAT3sdAbs were able to inhibit theinduction of the reporter cell line by interleukin-6, the activity ofthe two anti-STAT3 sdAbs were evaluated in a STAT3 reporter Thaw and UseGloResponse™ cell assay from Promega (Madison, Wis.).

Anti-STAT3 VHH13 (SEQ ID NO:3) sdAb, anti-STAT3 VHH14 (SEQ ID NO:4)sdAb, anti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb, and BB1608 (napabucasin)(Selleckchem, Houston, Tex.) were used. The concentration used for theassay was at 55.8 and 10μg/mL for anti-STAT3 VHH13 (SEQ ID NO:3) sdAb,84.3 and 10μg/mL for anti-STAT3 VHH14 (SEQ ID NO:4) sdAb, 50.5 and10μg/mL for anti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb and 4.16 and 2 μMfor BB1608.

The antibody blocking protocol provided with the Thaw and UseGloResponse™ SIE-luc2P/HEK293 cells was utilized with somemodifications. The modifications consisted of pretreating the cells withtest article for 48 or 24 hours prior to induction with IL-6.

The assay was performed over 4 days. On assay day 1, GloResponse™SIE-luc2P/HEK 293 cells were thawed and dispensed into 96-well plates at1×10⁴ cells/well. The sdAbs and BB1608 control were prepared in assaymedium (DMEM supplemented with 10% FBS) and added to triplicate wells.Media was added to wells designated for IL-6 positive and negativecontrols. All plates were then incubated at 37° C. with 5% CO₂ humidity.On assay day 2, an identical set of test article dilutions to day 1 wereprepared and added to triplicate wells. Media was added to wellsdesignated for IL-6 positive and negative controls.

IL-6 induction occurred on assay day 3. A 10× IL-6 stock (ThermoFisher,Hanover Park, Ill.) at the EC₈₀ concentration (40 ng/mL finalconcentration) was prepared in assay medium and added to the appropriatetest article and IL-6 control (“+IL6”) wells on each plate. A triplicateset of wells were designated as “−IL-6” and received assay media only.Plates were again incubated at 37° C. with 5% CO₂ humidity.

Detection was performed utilizing the Promega Bio-Glo™ Luciferase AssaySystem. On assay day 4, the Bio-Glo™ reagent was reconstituted per themanufacturer's instructions. At the end of induction, the plates wereremoved from the incubator and allowed to equilibrate to ambienttemperature for 10-15 minutes. The Bio-Glo™ Reagent was then added toeach well. After approximately 3-5 minutes, the relative luminescenceunits (RLU) was measured using the Veritas Microplate Luminator (TurnerBiosystems, Sunnyvale, Calif.).

Statistical analysis by ANOVA with Dunnett Multiple Comparisons Testusing the +IL6 as the control column was performed. The Pierce BCAProtein Assay Kit (Thermo Scientific, Rockford, Ill.) was utilized todetermine protein concentration. Cells were plated and treated with testarticle and IL-6 in parallel with the reporter assay. On assay day 4,cells were lysed and harvested for protein concentration utilizing theBCA kit.

Data from the STAT3 Reporter Assay and the BCA protein concentrationresults are reported in Tables 51 and 52. STAT3 Reporter Assay resultswere reported as a RLU value. The values were not normalized to proteinconcentration, although at the highest level of anti-STAT3 VHH13 (SEQ IDNO:3) sdAb, a decrease in protein concentration was observed.

TABLE 51 STAT3 Reporter Assay, 24 Hour Treatment Prior to IL6 InductionVHH13 VHH13 VHH14 VHH14 BB1608 BB1608 VHH69 VHH69 55.8 10 84.3 10 4.16 250.5 10 −IL6 +IL6 μg/mL μg/mL μg/mL μg/mL μM μM μg/mL μg/mL MediaLuminescence 359 132340 11445 169890 520343 203210 28 33 194320 18436419 (RLU) Luminescence 604 175040 12058 191537 503880 223150 31 39 198330165776 27 (RLU) Luminescence 489 174030 10992 164397 465649 218125 41 41203139 159115 31 (RLU) Average RLU 483 160470 11498 175275 496624 21482833 38 198596 169752 26 Standard 123 24367 535 14349 28060 10371 7 4 441613086 6 Deviation % CV 25 15 5 8 6 5 20 11 2 8 24 Average RLU 457 16044411473 175249 496598 214803 8 12 198571 169726 N/A with Media BlankFold-Induction 351 25 383 1086 470 0 0 434 371 N/A RLU/RLU of−IL6Protein 252 270 180 277 270 189 174 425 213 237 N/A concentration

TABLE 52 STAT3 Reporter Assay, 48 Hour Treatment Prior to IL6 InductionVHH13 VHH13 VHH14 VHH14 BB1608 BB1608 VHH69 VHH69 55.8 10 84.3 10 4.16 250.5 10 −IL6 +IL6 μg/mL μg/mL μg/mL μg/mL μM μM μg/mL μg/mL MediaLuminescence 634 172542 104 168740 494706 241785 25 26 237816 191241 33(RLU) Luminescence 476 158914 96 188307 488307 236843 31 42 220259171796 28 (RLU) Luminescence 485 169797 68 189606 458771 218982 32 31219104 155927 37 (RLU) Average RLU 532 167084 89 182218 480595 232537 2933 225726 172988 33 Standard 89 7208 19 11690 19169 11996 4 8 1048617687 5 Deviation % CV 17 4 21 6 4 5 13 25 5 10 14 Average RLU 499167052 57 182185 480562 232504 −3 0 225694 172955 N/A with Media BlankFold-Induction 335 0 365 963 466 0 0 452 347 N/A RLU/RLU of−IL6 Protein211 199 92 223 229 216 165 324 208 222 N/A concentration

Data was analyzed using an ANOVA. The data was shown to be statisticallysignificant at both 24 and 48 hour treatment time points; a P value of<0.0001 was obtained. Inhibition of IL-6 was seen in the wells treatedwith the highest concentration of anti-STAT3 VHH13 (SEQ ID NO:3) sdAband with the positive control (BB1608) with both the 24 hours and 48hours treatment (P values<0.01). Some of the lower RLU value with thehighest concentration of anti-STAT3 VHH13 (SEQ ID NO:3) sdAb can beattributed to the decrease in cell numbers (based on protein analysis).The decrease in protein is approximately 2-fold while the decrease inRLU is greater than 10-fold at 24 hours and greater than 1,000-fold at48 hours. Both concentrations of anti-STAT3 VHH14 (SEQ ID NO:4) sdAb andthe highest concentrations of anti-TNF-alpha VHH69 (SEQ ID NO:46) sdAbshowed an enhancement of the IL-6 induction (P values<0.05 and <0.01).The results indicate that anti-STAT3 VHH13 (SEQ ID NO:3) sdAb can entera cell and can suppress IL-6 production, with results comparable to asmall molecule inhibitor of STAT3.

Example 25: Anti-STAT3 sdAb Suppresses IL-6 Production

Anti-STAT3 VHH13 (SEQ ID NO:3) suppressed IL-6 production in HEp-2,MDA-MB-231, and PANC-1 cell lines. The cells were grown to confluence onchamber slides. Anti-STAT3 VHH13 (SEQ ID NO:3) sdAb was added toduplicate chambers (1:10 dilution in media) overnight in a 37° C.incubator. As a negative control, no antibody was added to duplicatechambers. The slides were washed, and either serum-free media alone or100 ng/ml recombinant human IL-6 (Peprotech, Rocky Hill, N.J.) in serumfree media was added to the slides for 15 min at 37° C. The media wasthen removed and the chamber slides were fixed in ice cold 100% methanolfor 10 min at −20° C.

Immunofluorescence assays were performed on the fixed slides. First, theslides were blocked with 3% BSA in PBS was done for one hour at roomtemperature. The primary antibody, Stat3(124H6) mouse monoclonalantibody (Cell Signaling Technology, Danvers, Mass.) was added to theslides at a 1:1000 dilution overnight in dark box at 4° C. The followingday, the slides were washed and the secondary Alexa Fluor 488 anti-mouseIgG antibody (Cell Signaling Technology) was added at a 1:500 dilutionand incubated for 1 hour at room temperature. The slides were thenwashed, mounted with mounting media and viewed with a fluorescencemicroscope.

Cells that were pre-treated with anti-STAT3 VHH13 (SEQ ID NO:3) sdAbalone showed Stat3 staining in the cytoplasm (FIG. 21 shows HEp-2 cells,but similar results were seen with MDA-MB-231 and PANC-1 cells). Thenucleus of cells treated with IL-6 stained positive for Stat3 (FIG. 22).The cells that were pre-treated with anti-STAT3 VHH13 (SEQ ID NO:3) sdAbfollowed by treatment with IL-6 did not stain positive for Stat3 in thenucleus, but rather showed Stat3 staining in the cytoplasm (FIG. 23).Thus, anti-STAT3 VHH13 (SEQ ID NO:3) sdAb blocks the translocation ofStat3 to the nucleus following IL-6 treatment.

Example 26: VEGF-A Analysis in Retinal Cells Treated with Anti-Stat3sdAb

Inhibition of the vascular endothelial growth factor A (VEGF-A)production was assessed in retinal cells exposed to anti-STAT3 sdAb, inparticular anti-STAT3 VHH13 (SEQ ID NO:3) sdAb. For Experiment 1, theretinal cell line ARPE-19 (ATCC, Manassas, Va.) were plated at 5×10⁴cells/well into a 24-well plate and grown to confluence. All incubationswith cells were performed in a 37° C., 5% CO₂ humidified incubator. Themedia was replaced with growth media (DMEM:HAM12) containing 1% FBS andincubated overnight. The media was then removed and replaced with mediacontaining anti-STAT3 VHH13 (SEQ ID NO:3) sdAb at 0.5 μg/mL, anti-STAT3VHH13 (SEQ ID NO:3) sdAb at 0.1 μg/ml, anti-STAT-3 monoclonal antibody(Abcam, Cambridge Mass.), or media alone, and incubated for 24 or 72hours. At that time, the supernatant was removed and stored at <−65° C.for ELISA analysis. Cells were lysed using RIPA lysis buffer(ThermoFisher Scientific, Rockford, Ill.) and the protein content of thecell lysate was measured in the BCA assay (ThermoFisher Scientific)according to the kit instructions. VEGF-A was measured utilizing theHuman VEGF-A ELISA Kit (ThermoFisher Scientific) in accordance with thekit instructions.

For Experiment 2, the assay was performed in a 96-well plate format.ARPE-19 cells were plated at 1.5×10⁴ cells/well into three 96-wellplates and allowed to grow to confluence. The media was replaced withgrowth media (DMEM:HAM12) containing 1% FBS and incubated overnight. Themedia was then replaced with media containing anti-STAT3 VHH13 (SEQ IDNO:3) sdAb at 100, 10, 1 or 0.1μg/ml, anti-EMP2 antibody (Abcam), ormedia, and incubated for 12, 24 or 48 hours. At the appropriate timepoint, the supernatant was removed and stored at ≦−65° C. for ELISAanalysis. Cells were lysed using RIM lysis buffer and the proteincontent of the cell lysate was measured in the BCA assay according tothe kit instructions. VEGF-A was measured utilizing the Human VEGF-AELISA Kit. For both experiments, statistical analysis by ANOVA withDunnett Multiple Comparisons Test using the negative control as thecontrol column was performed.

ELISA results were reported as pictograms (pg) VEGF-A/mL and thenadjusted for the dilution of samples in the ELISA (1:2 for Experiment 1and 1:5 for Experiment 2). The ELISA results were then normalized forprotein content. Data from Experiment 1 is reported in Table 53 andTable 54. For samples collected at 24 hours, inhibition of VEGF-A wasnot definitively detected. At 0.5 μg/mL anti-STAT3 VHH13 (SEQ ID NO:3)sdAb, triplicate wells ranged from 61.9 to 115 pg VEGF-A/m of proteinwhich was similar to the negative control (NC) media only wells whichranged from 59.3 to 105 pg VEFG/m protein. The positive control (PC)(anti-STAT3 monoclonal antibody) ranged from 48.8 to 66.4 pg VEFG/mprotein slightly lower but overlapping the range seen with the negativecontrol. Statistical analysis confirmed that the variation seen was notstatistically significant (P value>0.05). The samples collected at 72hours were higher than the top standard curve.

TABLE 53 Experiment 1 VEGF-A ELISA Results Result Mean Result Adj.Result* Mean % (pg VEGF-A (pg VEGF-A Std. (pg VEGF-A (pg VEGF-A Std.Negative Sample per mL) per mL) Dev. CV % per mL) per mL) Dev. ControlSTAT3 VHH13 at 0.5 466 637 241.6 37.9 1274 2664 1303.3 92.0 μg/well 24Hr Well 1 808 STAT3 VHH13 at 0.5 1277 1429 214.2 15.0 2857 μg/well 24 HrWell 2 1580 STAT3 VHH13 at 0.5 1823 1930 150.5 7.8 3859 μg/well 24 HrWell 3 2036 STAT3 VHH13 at 0.1 464 520 78.3 15.1 1040 2060 1280.0 71.1μg/well 24 Hr Well 1 575 STAT3 VHH13 at 0.1 1478 1748 1748 381.6 3496μg/well 24 Hr Well 2 2018 STAT3 VHH13 at 0.1 727 822 822 134.7 1644μg/well 24 Hr Well 3 917 PC (anti-STAT3 551 664 159.9 24.1 1328 1970556.6 68.0 antibody @ 1:500 777 dil) 24 Hr Well 1 PC (anti-STAT3 10821155 1155 103.2 2311 antibody @ 1:500 1228 dil) 24 Hr Well 2 PC(anti-STAT3 1086 1136 1136 70.6 2272 antibody @ 1:500 dil) 24 Hr Well 3NC (media only) 928 1008 113.5 11.3 2016 2896 826.1 100.0 24 Hr Well 1NC (media only) 1668 1828 1828 226.1 3655 24 Hr Well 2 NC (media only)1496 1508 1508 17.2 3016 24 Hr Well 3 STAT3 VHH13 at 0.5 >6,000 1289811481845.2 37.4 >12,000 μg/well 72 Hr Well 1 >6,000 STAT3 VHH13 at0.5 >6,000 320652 1461.5 0.5 >12,000 μg/well 72 Hr Well 2 >6,000 STAT3VHH13 at 0.5 >6,000 351430 166765.9 47.5 >12,000 μg/well 72 Hr Well3 >6,000 STAT3 VHH13 at 0.1 >6,000 896832 338049.2 37.7 >12,000 μg/well72 Hr Well 1 >6,000 STAT3 VHH13 at 0.1 >6,000 4936142 0.0 0.0 >12,000μg/well 72 Hr Well 2 >6,000 STAT3 VHH13 at 0.1 >6,000 282889 86269.830.5 >12,000 μg/well 72 Hr Well 3 >6,000 PC (anti-STAT3 >6,000 Range?Range? Range? >12,000 antibody @ 1:500 >6,000 dil) 72 Hr Well 1 PC(anti-STAT3 >6,000 10802 215.7 2.0 >12,000 antibody @ 1:500 >6,000 dil)72 Hr Well 2 PC (anti-STAT3 >6,000 Range? Range? Range? >12,000 antibody@ 1:500 >6,000 dil) 72 Hr Well 3 NC (media only) >6,000 45898 41321.190.0 >12,000 72 Hr Well 1 >6,000 NC (media only) 1065 3417 3326.5 97.36835 72 Hr Well 2 5769 NC (media only) >6,000 418930 170327.240.7 >12,000 72 Hr Well 3 >6,000 *All samples tested at a 1:2 dilution

TABLE 54 Experiment 1 VEGF-A Normalized Results Mean Protein Total pgVEGF-A (pg VEGF-A pg VEGF-A Conc. protein per μg per μg Std. % Sampleper well^(#) (μg/mL) per well^(#) protein protein) Dev. Control STAT3VHH13 at 0.5 637 206 10 62 99 41.7 115.8 μg/well 24 Hr Well 1 STAT3VHH13 at 0.5 1429 315 16 91 μg/well 24 Hr Well 2 STAT3 VHH13 at 0.5 1930268 13 144 μg/well 24 Hr Well 3 STAT3 VHH13 at 0.1 520 245 12 42 66 3776.8 μg/well 24 Hr Well 1 STAT3 VHH13 at 0.1 1748 323 16 108 μg/well 24Hr Well 2 STAT3 VHH13 at 0.1 822 357 18 46 μg/well 24 Hr Well 3 PC(anti-STAT3 664 272 14 49 58 8.9 68.4 antibody @ 1:500 dil) 24 Hr Well 1PC (anti-STAT3 1155 387 19 60 antibody @ 1:500 dil) 24 Hr Well 2 PC(anti-STAT3 1136 342 17 66 antibody @ 1:500 dil) 24 Hr Well 3 NC (mediaonly) 1008 340 17 59 85 23.5 100.0 24 Hr Well 1 NC (media only) 1828 34817 105 24 Hr Well 2 NC (media only) 1508 329 16 92 24 Hr Well 3^(#)Based on collecting 0.5 ml of supernatant per well and the additionof 0.05 μl per well of RIPA buffer.

Data from Experiment 2 is reported in Table 55 and Table 56. Data wasanalyzed using an ANOVA. The data was shown to be statisticallysignificant at all time points. For the 12 and 48 hour treatments a Pvalue of <0.0001 was obtained and for the 24 hour treatment the P valuewas 0.0004. At 100 μg/mL anti-STAT3 VHH13, less VEGF-A was detected. At48 hours, the pg VEGF-A/m protein for the 100μg/mL anti-STAT3 VHH13treatment was at 11% of the negative control (P<0.01). Similar decreasesin VEGF-A were seen with the 12 and 24 hour treatments (20.1% and 7.5%decrease respectively; P values of <0.01 for both). This decrease inVEGF-A was not seen with lower concentrations of anti-STAT3 VHH13. Itshould be noted that less protein was also detected from the wells with100μg/mL anti-STAT3 VHH13, which could be due to the inhibition of cellgrowth and proliferation, resulting in the lower concentrations ofprotein seen in Experiment 2.

TABLE 55 Experiment 2 VEGF-A ELISA Results Result Mean Result Adj.Result* Mean % (pg VEGF-A (pg VEGF-A Std. CV (pg VEGF-A (pg VEGF-A Std.Negative Sample per mL) per mL) Dev. % per mL) per mL) Dev. ControlSTAT3 VHH13 at 0.1 29.44 28.54 1.272 4.5 142.71 159.7 23.13 123.8μg/well 12 Hr Well 1 27.64 STAT3 VHH13 at 0.1 29.74 30.06 0.455 1.5150.31 μg/well 12 Hr Well 2 30.38 STAT3 VHH13 at 0.1 36.84 37.2 0.5191.4 186.02 μg/well 12 Hr Well 3 37.57 STAT3 VHH13 at 1 26.74 25.82 1.3 5129.12 142.5 16.67 110.5 μg/well 12 Hr Well 1 24.91 STAT3 VHH13 at 126.62 27.43 1.15 4.2 137.14 μg/well 12 Hr Well 2 28.24 STAT3 VHH13 at 133.18 32.23 1.337 4.1 161.16 μg/well 12 Hr Well 3 31.29 STAT3 VHH13 at10 29.66 30.43 1.092 3.6 152.14 139.00 15.4 107.8 μg/well 12 Hr Well 131.2 STAT3 VHH13 at 10 24.52 24.41 0.151 0.6 122.07 μg/well 12 Hr Well 224.31 STAT3 VHH13 at 10 27.98 28.58 0.848 3 142.92 μg/well 12 Hr Well 329.18 STAT3 VHH13 at 100 5.09 4.86 0.327 6.7 24.27 23.8 0.79 18.5μg/well 12 Hr Well 1 4.62 STAT3 VHH13 at 100 5.3 4.86 0.623 12.8 24.27μg/well 12 Hr Well 2 4.41 STAT3 VHH13 at 100 4.83 4.58 0.356 7.8 22.91μg/well 12 Hr Well 3 4.33 NC (media only) 28.76 26.76 0 0 143.78 128.923.87 100 12 Hr Well 1 28.76 NC (media only) 28.58 28.33 0.363 1.3141.63 12 Hr Well 2 28.07 NC (media only) 20.52 20.28 0.311 1.6 101.4 12Hr Well 3 20.05 PC (anti-STAT3 25.76 25.31 0.634 2.5 12656 120.1 12.6593.2 antibody @ 1:500 24.86 dil) 12 Hr Well 1 PC (anti-STAT3 20.77 21.110.482 2.3 105.55 antibody @ 1:500 21.45 dil) 12 Hr Well 2 PC (anti-STAT328.33 25.66 3.777 14.7 128.28 antibody @ 1:500 22.99 dil) 12 Hr Well 3STAT3 VHH13 at 0.1 58.86 55.73 4.431 8 278.66 287.4 14 93.6 μg/well 24Hr Well 1 52.60 STAT3 VHH13 at 0.1 59.52 56.02 4.959 8.9 280.09 μg/well24 Hr Well 2 52.51 STAT3 VHH13 at 0.1 64.54 60.72 5.413 8.9 303.58μg/well 24 Hr Well 3 56.89 STAT3 VHH13 at 1 78.44 74.61 5.413 7.3 373.06327.9 39.69 106.8 μg/well 24 Hr Well 1 70.78 STAT3 VHH13 at 1 68.6162.42 8.755 14 312.11 μg/well 24 Hr Well 2 56.23 STAT3 VHH13 at 1 63.7159.71 5.658 9.5 298.74 μg/well 24 Hr Well 3 55.71 STAT3 VHH13 at 1057.85 55.95 2.696 4.8 279.74 350.7 72.26 1142 μg/well 24 Hr Well 1 54.04STAT3 VHH13 at 10 72.69 69.61 4.356 6.3 348.06 μg/well 24 Hr Well 266.53 STAT3 VHH13 at 10 84.97 84.84 0.19 0.2 424.20 μg/well 24 Hr Well 384.71 STAT3 VHH13 at 100 6.01 5.84 0.238 4.1 29.21 19.2 8.66 6.3 μg/well24 Hr Well 1 5.67 STAT3 VHH13 at 100 2.82 2.86 0.059 2.1 14.31 μg/well24 Hr Well 2 2.9 STAT3 VHH13 at 100 2.61 2.82 0.296 10.5 14.10 μg/well24 Hr Well 3 3.03 NC (media only) 62.91 62.21 0.997 1.6 311.04 307.145.18 100 24 Hr Well 1 61.5 NC (media only) 51.81 52.01 0.278 0.5 260.0424 Hr Well 2 52.21 NC (media only) 70.61 70.03 0.815 1.2 350.15 24 HrWell 3 69.45 PC (anti-STAT3 48.11 45.11 4.238 9.4 225.54 264.1 8.66 6.3antibody @ 1:500 42.11 dil) 24 Hr Well 1 PC (anti-STAT3 55.27 51.994.633 8.9 259.95 antibody @ 1:500 48.72 dil) 24 Hr Well 2 PC (anti-STAT359.44 61.35 2.708 4.4 306.75 antibody @ 1:500 63.27 dil) 24 Hr Well 3STAT3 VHH13 at 0.1 195.75 189.30 9.122 4.8 946.50 966 45.18 100 μg/well48 Hr Well 1 182.85 STAT3 VHH13 at 0.1 173.80 170.27 4.997 2.9 851.33μg/well 48 Hr Well 2 166.73 STAT3 VHH13 at 0.1 220.87 220.04 1.17 0.51100.22 μg/well 48 Hr Well 3 219.22 STAT3 VHH13 at 1 168.84 175.22 9.0125.1 876.08 1004.7 40.76 86 μg/well 48 Hr Well 1 181.59 STAT3 VHH13 at 1195.75 203.89 11.508 5.6 1019.44 μg/well 48 Hr Well 2 212.03 STAT3 VHH13at 1 225.75 223.71 2.880 1.3 1118.55 μg/well 48 Hr Well 3 221.67 STAT3VHH13 at 10 194.18 191.17 4.257 2.2 955.85 925.1 125.59 99.3 μg/well 48Hr Well 1 188.16 STAT3 VHH13 at 10 194.77 196.56 2.538 1.3 982.81μg/well 48 Hr Well 2 198.36 STAT3 VHH13 at 10 158.85 167.34 12.002 7.2836.69 μg/well 48 Hr Well 3 175.83 STAT3 VHH13 at 100 7.83 7.78 0.0821.1 38.88 35.6 5.54 3.7 μg/well 48 Hr Well 1 7.72 STAT3 VHH13 at 1007.49 7.76 0.384 5 38.78 μg/well 48 Hr Well 2 8.03 STAT3 VHH13 at 1005.86 5.85 0.027 0.5 29.23 μg/well 48 Hr Well 3 5.83 NC (media only)202.96 195.55 10.455 5.3 977.76 972.4 32.87 100 48 Hr Well 1 188.16 NC(media only) 209.84 200.46 13.254 6.6 1002.32 48 Hr Well 2 191.09 NC(media only) 205.67 187.45 25.767 13.7 937.23 48 Hr Well 3 169.23 PC(anti-STAT3 186.06 177.48 12.142 6.8 887.38 887.7 41.95 91.3 antibody @1:500 168.89 dil) 48 Hr Well 1 PC (anti-STAT3 196.49 185.96 14.884 8929.82 antibody @ 1:500 175.44 dil) 48 Hr Well 2 PC (anti-STAT3 177.32169.18 11.512 6.8 845.90 antibody @ 1:500 161.04 dil) 48 Hr Well 3 *Allsamples tested at a 1:2 dilution

TABLE 56 Experiment 2 VEGF-A Normalized Results Mean Protein Total pgVEGF-A (pg VEGF-A pg VEGF-A Conc. ML RIPA protein per μg per μg Std. %Sample per mL^(#) (μg/mL) buffer/well per well protein protein) Dev.Control STAT3 VHH13 at 0.1 14.3 67 0.1 6.7 2.1 2.540 0.5 134.4 μg/well12 Hr Well 1 STAT3 VHH13 at 0.1 15 62 0.1 6.2 2.4 μg/well 12 Hr Well 2STAT3 VHH13 at 0.1 18.6 61 0.1 6.1 3 μg/well 12 Hr Well 3 STAT3 VHH13 at1 12.9 66 0.1 6.6 2 2.273 0.4 120.3 μg/well 12 Hr Well 1 STAT3 VHH13 at1 13.7 63 0.1 6.3 2.2 μg/well 12 Hr Well 2 STAT3 VHH13 at 1 16.1 60 0.16 2.7 μg/well 12 Hr Well 3 STAT3 VHH13 at 10 15.2 61 0.1 6.1 2.5 2.1480.3 113.7 μg/well 12 Hr Well 1 STAT3 VHH13 at 10 12.2 64 0.1 6.4 1.9μg/well 12 Hr Well 2 STAT3 VHH13 at 10 14.3 70 0.1 7 2 μg/well 12 HrWell 3 STAT3 VHH13 at 100 2.4 64 0.1 6.4 0.4 0.4 0.380 0 μg/well 12 HrWell 1 STAT3 VHH13 at 100 2.4 62 0.1 6.2 0.4 μg/well 12 Hr Well 2 STAT3VHH13 at 100 2.3 62 0.1 6.2 0.4 μg/well 12 Hr Well 3 NC (media only)14.4 73 0.1 7.3 2 1.889 0.3 100 12 Hr Well 1 NC (media only) 14.2 67 0.16.7 2.1 12 Hr Well 2 NC (media only) 10.1 64 0.1 6.4 1.6 12 Hr Well 3 PC(anti-STAT3 12.7 66 0.1 6.6 1.9 1.917 0.2 101.5 antibody @ 1:500 dil) 12Hr Well 1 PC (anti-STAT3 10.6 61 0.1 6.1 1.7 antibody @ 1:500 dil) 12 HrWell 2 PC (anti-STAT3 12.8 61 0.1 6.1 2.1 antibody @ 1:500 dil) 12 HrWell 3 STAT3 VHH13 at 0.1 27.9 83 0.089 7.4 3.8 4.152 0.5 94.1 μg/well24 Hr Well 1 STAT3 VHH13 at 0.1 28 72 0.097 7.0 4.0 μg/well 24 Hr Well 2STAT3 VHH13 at 0.1 30.4 73 0.089 6.5 4.7 μg/well 24 Hr Well 3 STAT3VHH13 at 1 37.3 68 0.1 6.8 5.5 4.084 1.6 92.5 μg/well 24 Hr Well 1 STAT3VHH13 at 1 31.2 71 0.1 7.1 4.4 μg/well 24 Hr Well 2 STAT3 VHH13 at 129.9 126 0.1 12.6 2.4 μg/well 24 Hr Well 3 STAT3 VHH13 at 10 28 64 0.16.4 4.4 5.290 0.9 119.8 μg/well 24 Hr Well 1 STAT3 VHH13 at 10 34.8 640.1 6.4 5.4 μg/well 24 Hr Well 2 STAT3 VHH13 at 10 42.4 70 0.1 7 6.1μg/well 24 Hr Well 3 STAT3 VHH13 at 100 2.9 62 0.1 6.2 0.5 0.329 0.1 7.5μg/well 24 Hr Well 1 STAT3 VHH13 at 100 1.4 56 0.1 5.6 0.3 μg/well 24 HrWell 2 STAT3 VHH13 at 100 1.4 54 0.1 5.4 0.3 μg/well 24 Hr Well 3 NC(media only) 31.1 76 0.095 7.2 4.3 4.414 1.1 100 24 Hr Well 1 NC (mediaonly) 26 77 0.1 7.7 3.4 24 Hr Well 2 NC (media only) 35 105 0.06 6.3 5.624 Hr Well 3 PC (anti-STAT3 22.6 73 0.087 6.4 3.6 3.838 0.3 86.9antibody @ 1:500 dil) 24 Hr Well 1 PC (anti-STAT3 26 72 0.096 6.9 3.8antibody @ 1:500 dil) 24 Hr Well 2 PC (anti-STAT3 30.7 73 0.1 7.3 4.2antibody @ 1:500 dil) 24 Hr Well 3 STAT3 VHH13 at 0.1 94.7 74 0.091 6.714.1 14.067 1 107.4 μg/well 48 Hr Well 1 STAT3 VHH13 at 0.1 85.1 740.088 6.5 13.1 μg/well 48 Hr Well 2 STAT3 VHH13 at 0.1 110 73 0.1 7.315.1 μg/well 48 Hr Well 3 STAT3 VHH13 at 1 87.6 72 0.098 7.1 12.4 13.7961.5 105.4 μg/well 48 Hr Well 1 STAT3 VHH13 at 1 101.9 77 0.097 7.5 13.6μg/well 48 Hr Well 2 STAT3 VHH13 at 1 111.9 73 0.1 7.3 15.3 μg/well 48Hr Well 3 STAT3 VHH13 at 10 95.6 71 6.8 6.8 14 13.696 1.8 104.6 μg/well48 Hr Well 1 STAT3 VHH13 at 10 98.3 67 6.4 6.4 15.3 μg/well 48 Hr Well 2STAT3 VHH13 at 10 83.7 71 701 7.1 11.8 μg/well 48 Hr Well 3 STAT3 VHH13at 100 3.9 27 2.7 2.7 1.4 1.458 0.1 11.1 μg/well 48 Hr Well 1 STAT3VHH13 at 100 3.9 25 2.5 2.5 1.6 μg/well 48 Hr Well 2 STAT3 VHH13 at 1003.9 22 2.1 2.1 1.4 μg/well 48 Hr Well 3 NC (media only) 97.8 75 7.5 7.513 13.092 0.6 100 48 Hr Well 1 NC (media only) 100.2 83 7.3 7.3 13.7 48Hr Well 2 NC (media only) 93.7 78 7.5 7.5 12.5 48 Hr Well 3 PC(anti-STAT3 88.7 69 6.8 6.8 131 13.412 1 102.4 antibody @ 1:500 dil) 48Hr Well 1 PC (anti-STAT3 93 68 6.4 6.4 14.5 antibody @ 1:500 dil) 48 HrWell 2 PC (anti-STAT3 84.6 68 6.7 6.7 12.6 antibody @ 1:500 dil) 48 HrWell 3 ^(#)Based on collecting 0.1 ml of supernatant.

The treatment of retinal cells with 100 μg/mL of anti-STAT3 VHH13 (SEQID NO:3) sdAb decreased the production of VEGF-A. This may be associatedwith the inhibition of STAT3 and subsequent effect on the growth andproliferation of the retinal cells.

Example 27: Anti-STAT3 VHH13 (SEQ ID NO:3) sdAb Specificity

Experiments were performed to assess the specificity of multiple STATsdAbs including STAT3 sdAb and the STAT5 sdAb bind to other STATproteins using an ELISA format. A panel of STAT proteins (STAT 1, STAT2, STAT 3, STAT 4, STAT 5a, STAT 5b and STAT 6) were used.

The test articles for this study were STAT 3 VHH labeled ProcessIntermediate III from Anthem Biosciences (Bangalore, India), anti-STAT3VHH13 (SEQ ID NO:3) sdAb, and STAT5-31 (SEQ ID NO:83) sdAb. The testarticles were tested at 10, 1, 0.1 and 0.01 μg/mL.

A standard sandwich ELISA format was utilized. For each ELISA, wells ona 96-well plate were coated with one of the STAT proteins at 0.5 μg/mLin carbonate buffer. Plates were incubated overnight at 2-8° C. Afterthe incubation, plates were washed (3×) and blocked (3% BSA in PBS) forat least one hour at room temperature.

After removal of the blocking buffer and washing (3×), samples wereadded to the appropriate wells of the ELISA plates and incubated at roomtemperature with shaking (150 rpm) for at least one hour. The secondaryantibody was then added to each well and incubated with shaking for atleast one hour at room temperature. On Run 1, the secondary antibody wasrabbit anti-llama biotinylated antigen at 1:5,000 dilution. For Run 2,in addition to the rabbit anti-lama, the his-probe (H-3) biotin labeledsecondary antibody at a 1:1,000 dilution was tested in parallel.

After the incubation with secondary antibody, the plates were againwashed (3×) and horseradish peroxidase (HRP) at a 1:25,000 dilution wasadded to each well. Plates were incubated with shaking for at least onehour at room temperature. One last wash (3×) was performed followed bythe addition of TMB (Tetramethylbenzidine) substrate. After a 20 to 22minute incubation at room temperature, stop solution was added to haltthe colorimetric reaction. The absorbance at 450 nm was measured on aVersaMax instrument utilizing SoftMax Pro GxP version 5.4 software fromMolecular Devices.

The STAT3 VHH from Anthem, anti-STAT3 VHH13 (SEQ ID NO:3) sdAb, andSTAT5-31 (SEQ ID NO:83) sdAb were detected in each of the STAT 1, STAT2, STAT 3, STAT 4, STAT 5a, STAT 5b and STAT 6 ELISA. Mean OD₄₅₀ valuesare shown in Table 57. The STAT 3 VHH from Anthem showed a higherspecify to STAT 3 as indicated by a higher OD value as compared with theother ELISAs. The anti-STAT3 VHH13 (SEQ ID NO:3) sdAb had higher valuesin the STAT 3 and STAT 4 ELISA. The STAT5-31 (SEQ ID NO:83) sdAb hadhigher values in both the STAT 5a and the STAT 1 ELISA; however, theoverall values seen with the STAT5-31 (SEQ ID NO:83) sdAb wereconsiderably lower than those seen with the anti-STAT3 VHH13 (SEQ IDNO:3) sdAb in the same ELISAs. While it was demonstrated that these VHHsare not specific for their targeted STAT some preferential specificitywas shown.

TABLE 57 STAT Specificity Mean OD450 Value* Concentration Sample μg/mLSTAT 1 STAT 2 STAT 3 STAT 4 STAT 5a STAT 5b STAT 6 STAT 3 VHH 0.01 0.0080.007 0.007 0.008 0.005 0.008 0.007 from Anthem 0.1 0.009 0.007 0.0450.020 0.020 0.023 0.019 1 0.203 0.270 0.776 0.237 0.250 0.264 0.261 101.990 2.266 3.243 2.246 2.153 2.354 2.265 STAT 3 VHH13 0.01 0.026 0.0230.063 0.083 0.107 0.038 0.044 0.1 0.159 0.186 0.431 0.833 0.821 0.4990.413 1 1.938 1.846 2.759 3.140 2.725 2.297 2.094 10 3.163 3.336 3.6693.674 3.322 3.377 3.205 STAT 5-31 0.01 0.024 0.007 0.009 0.006 0.0170.005 0.009 0.1 0.015 0.000 0.000 0.000 0.000 0.000 0.000 1 0.056 0.0150.014 0.012 0.000 0.009 0.007 10 0.122 0.076 0.088 0.085 0.158 0.0510.048 *Mean OD₄₅₀ values from duplicate wells

The results show that the STAT3 VHH from Anthem and anti-STAT3 VHH13(SEQ ID NO:3) sdAb bind to STAT proteins with an increased affinity forthe STAT3 protein. The STAT5-31 (SEQ ID NO:83) sdAb binds to STAT 1,STAT 2, STAT 3, STAT 4, STAT 5a, STAT 5b and STAT 6 proteins with anincreased affinity for the STAT 5a protein.

Example 28: Anti-TNF-Alpha sdAbs Bind to TNF-Alpha Protein

In this Example, the binding of anti-TNF-α sdAbs to active human TNF-αfull length protein was assessed using an enzyme linked immunosorbentassay (ELISA) format. Additionally, the feasibility of utilizing asecondary antibody targeted to the his tag portion of the sdAbs wasassessed.

The test articles for this study were anti-TNF-alpha VHH62 (SEQ IDNO:47) sdAb, TNF-alpha VHH66 (SEQ ID NO:45) sdAb, and anti-TNF-alphaVHH69 (SEQ ID NO:46) sdAb (Experiment 1). Subsequently, additional lotsof anti-TNF-alpha VHH62 (SEQ ID NO:47) sdAb and VHH69 (Experiment 4), aswell as anti-TNF-alpha VHH66 (SEQ ID NO:45) sdAb (Experiment 5) werereceived. For the testing of the secondary antigen (Experiments 2 and3), the following sdAb molecules were included: anti-STAT3 VHH13 (SEQ IDNO:3) sdAb, anti-STAT3 VHH14 (SEQ ID NO:4) sdAbs, anti-KRAS (G12D) (SEQID NO:2) sdAb, and a commercially available anti-Stat3 antibody (Abcam,Cambridge, Mass.).

For the assays to assess the binding of the sdAbs to TNF-alpha protein(Experiment 1, 4 and 5), a standard sandwich ELISA format was utilized.For each ELISA, wells on a 96-well plate were coated with TNF proteins(Abcam, Cambridge, Mass.) at 0.5, 0.25 or 0.125 μg/mL in carbonatebuffer for the initial experiment and at 0.5 μg/mL only for subsequentassays. Plates were incubated overnight at 2-8° C. After the incubation,plates were washed (3×) and blocked (3% Bovine Serum Albumin (BSA) inPhosphate Buffered Saline (PBS)) for at least one hour at roomtemperature.

After removal of the blocking buffer and washing (3×), samples wereadded to the appropriate wells of the ELISA plates and incubated at roomtemperature with shaking (150 rpm) for at least one hour. The secondaryantibody (rabbit anti-llama biotinylated antibody at either a 1:5,000 or10,000 dilution) was then added to each well and incubated with shakingfor at least one hour at room temperature.

After the incubation with secondary antibody, the plates were againwashed (3×) and horseradish peroxidase (HRP) at either a 1:25,000 or1:50,000 dilution was added to each well. Plates were incubated withshaking for at least one hour at room temperature. A final wash (3×) wasperformed followed by the addition of TMB (Tetramethylbenzidine)substrate. After a 20 to 25 minute incubation at room temperature, stopsolution was added to halt the colorimetric reaction. The absorbance at450 nm was measured on a VersaMax instrument utilizing SoftMax Pro GxPversion 5.4 software from Molecular Devices (Sunnyvale, Calif.).

Two assays (Experiment 2 and 3) were performed to assess the binding ofthe secondary antibody to the sdAbs. A similar ELISA method was utilizedexcept the sdAbs were directly coated onto the assay plate at aconcentration of 1μg/mL. Plates were incubated overnight at 2-8° C.After the incubation, plates were washed (3×) and blocked (3% BSA inPBS) for at least one hour at room temperature.

After removal of the blocking buffer and washing (3×), the secondaryantibodies, either rabbit anti-llama biotinylated antibody or his-probe(H-3) biotin labeled antibody at was then added at a 1:5,000 dilution toeach well and incubated with shaking for at least one hour at roomtemperature. Only the rabbit anti-llama biotinylated antibody was usedin Experiment 2. Both antibodies were run in parallel in Experiment 3.

After the incubation with secondary antibody, the plates were againwashed (3×) and HRP at a 1:50,000 dilution was added to each well.Plates were incubated with shaking for at least one hour at roomtemperature. One last wash (3×) was performed followed by the additionof TMB substrate and plates were incubated at room temperature. For theanti-llama biotinylated antibody stop solution was added to halt thecolorimetric reaction at 10 minutes. The his-probe (H-3) biotin labeledantibody was allowed to incubate for 20 minutes prior to stopping. Theabsorbance at 450 nm was measured as before.

For Experiment 1, a checkerboard of coating antigen concentration (0.5,0.25, and 0.125 μg/mL), secondary antibody (1:5,000 and 1:10,000) andHRP (1:25,000 and 1:50,000) were assessed for signal to noise against 1μg/mL of each sdAb. Signal/Noise ratios are provided in Table 58 foranti-TNF-alpha VHH62 (SEQ ID NO:47) sdAb, anti-TNF-alpha VHH66 (SEQ IDNO:45) sdAb and anti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb respectively.Both anti-TNF-alpha VHH62 (SEQ ID NO:47) sdAb and anti-TNF-alpha VHH69(SEQ ID NO:46) sdAb had little to no detection (i.e. very low OD values)indicating poor binding of the sdAbs to the TNF-α protein or to therabbit anti-Llama secondary antibody. The anti-TNF-alpha VHH66 (SEQ IDNO:45) sdAb demonstrated good binding to the TNF-α protein with signalto noise ratios as high as 44.8.

TABLE 58 TNF ELISA Signal/Noise Ratio Experiment 1 Signal/Noise Ratioanti- Rabbit TNF-alpha TNF-alpha TNF-alpha anti-Llama VHH62 VHH66 VHH69Coating Secondary (SEQ ID (SEQ ID (SEQ ID antigen Antibody HRP (1:X NO:47) NO: 45) NO: 46) (μg/mL) (1:X dilution) dilution) sdAb sdAb sdAb 0.55,000 25,000 2.0 40.4 8.0 0.5 5,000 50,000 1.9 44.8 7.5 0.5 10,00025,000 1.7 41.9 5.9 0.5 10,000 50,000 1.5 32.4 3.7 0.25 5,000 25,000 1.535.3 3.2 0.25 5,000 50,000 1.4 28.7 2.4 0.25 10,000 25,000 1.4 25.7 2.10.25 10,000 50,000 1.3 18.5 1.9 0.125 5,000 25,000 1.1 11.8 1.6 0.1255,000 50,000 1.1 12.4 1.6 0.125 10,000 25,000 1.1 11.4 1.5 0.125 10,00050,000 0.9 7.3 1.3

The binding of the secondary antibody was assessed in Experiments 2 and3 to determine if the lack of detection with anti-TNF-alpha VHH62 (SEQID NO:47) sdAb and anti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb was due tobinding to the coating antigen or to the secondary antibody. Data isshown in Table 59 below. Additional VHH molecules were included in theseELISAs as indicated. In Experiment 2, high OD₄₅₀ values (>1) wereobtained with all but the anti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb. Thisindicates that the rabbit anti-llama Secondary Antibody is anappropriate antibody for these ELISAs. For Experiment 3, an anti-hissecondary antibody was compared to the anti-llama secondary antibody.For all but one sdAb, anti-STAT3 VHH14 (SEQ ID NO:4), lower OD₄₅₀ valueswere obtained with the anti-his secondary antibody.

A comparison of data obtained between Experiment 2 and Experiment 3showed a decrease in OD values for several of the sdAbs tested. ForExperiment 3 the 1:100 dilution of sdAb that had been prepared forExperiment 2 was utilized instead of preparing a fresh dilution (thiswas done to conserve material as several were in limited supply). The1:100 dilution had been prepared in PBS and stored at −20° C. for threemonths. The decrease in OD₄₅₀ values is an indication of samplestability. Either storing the dilute solution and/or freeze thawing thesamples resulted in the decreased stability.

TABLE 59 Comparison of Secondary Biotinylated Antibodies in Experiments2 and 3 OD₄₅₀ Values Rabbit anti-Llama Rabbit anti-Llama His-probeSecondary Antibody Secondary Antibody (H-3) Sample Experiment 2Experiment 3 Experiment 3 Anti-STAT3 VHH14 (SEQ ID NO: 4) sdAb 2.8132.439 3.416 Anti-KRAS (G12D) (SEQ ID NO: 2) sdAb 1.924 1.307 0.577Anti-STAT3 VHH13 (SEQ ID NO: 3) sdAb 1.313 0.363 0.360 Anti-TNF-alphaVHH62 (SEQ ID NO: 47) sdAb 1.485 2.341 0.196 Anti-TNF-alpha VHH66 (SEQID NO: 45) sdAb 2.057 2.682 0.181 Anti-TNF-alpha VHH69 (SEQ ID NO: 46)sdAb 0.499 0.443 0.153 Anti-Stat 3 commercial antibody Not tested 3.0760.018□□ {circle around (1)}Documentation does not indicate that this VHHhas a his-tag.

New batches of anti-TNF-alpha VHH62 (SEQ ID NO:47) sdAb andanti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb were tested using four 10-foldserial dilutions starting at 1 μg/mL. Again very little detection wasseen with the anti-TNF-alpha VHH62 (SEQ ID NO:47) sdAb (data not shown).The original receipt performed better than the new receipt; however, ODvalues were poor (<0.2 OD₄₅₀). For anti-TNF-alpha VHH69 (SEQ ID NO:46)sdAb, sufficient OD values were obtained to demonstrate a dose responseof OD₄₅₀ to concentration of sdAb, as shown in FIG. 22. A comparison ofthe old and new lot of anti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb showedsimilar results with the new batch having slightly higher OD values.

Two new batches of anti-TNF-alpha VHH66 (SEQ ID NO:45) sdAb were testedin Experiment 5 alongside the original batch. Each sdAb was tested atfour 10-fold dilutions starting at 1 μg/mL. Similar detection was seenwith all three batches (data not shown). Of the conditions tested, thecombination of 0.5 μg/mL coating antigen, secondary antibody at 1:5,000and HRP at 1:25,000 was shown to be optimal.

The final experiment performed was to confirm that the results from theELISA testing were due to binding to the TNF-α protein and notnon-specific binding of the sdAb. For this determination, a comparisonwas made between wells coated with 0.5 μg/mL of coating antigen andnon-coated wells. The results are provided in Table 60. As previouslyseen, 1 μg/mL of anti-TNF-alpha VHH62 (SEQ ID NO:47) sdAb was notdetected in either coated or non-coated wells. Both the anti-TNF-alphaVHH66 (SEQ ID NO:45) sdAb and anti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb(at 1μg/mL) were detected in coated wells but not in non-coated wells.This experiment confirms that the detection seen with the previousexperiments was due to the specific binding of the VHH to the coatingantigen and not due to non-specific binding.

TABLE 60 Assessment of Non-specific Binding OD450 Values anti-TNF-alphaTNF-alpha VHH62 VHH66 TNF-alpha VHH69 (SEQ ID NO: 47) (SEQ ID NO: 45)(SEQ ID NO: 46) sdAb sdAb sdAb Coated 0.005 3.863 1.948 Non-Coated−0.001 0.009 0.007

The results showed that both anti-TNF-alpha VHH66 (SEQ ID NO:45) sdAband anti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb demonstrated binding to theTNF-α protein. Detection of anti-TNF-alpha VHH62 (SEQ ID NO:47) sdAb wasnot demonstrated. Of the three anti-TNF-α sdAbs tested, anti-TNF-alphaVHH66 (SEQ ID NO:45) sdAb demonstrated the best affinity to the TNF-αprotein. Additional preparations of each sdAb demonstrated similarresults, indicating consistency between batch preparations.

Example 29: Cytotoxicity of Anti-TNF-α VHH66 sdAb (SEQ ID NO 45) on L929Fibroblast Cells

This Example demonstrates the anti-proliferative effects of theanti-TNF-α VHH66 (SEQ ID NO:45) sdAb. The activity of the anti-TNF-αVHH66 (SEQ ID NO:45) sdAb was assessed by incubating the anti-TNF-αVHH66 (SEQ ID NO:45) sdAb with a cytotoxic dose of TNFα, followed byevaluating the mixture for the inhibition of cytotoxicity in the TNFαsensitive mouse fibroblast cell line L929 (ATCC, Manassas, Va.).

Promega's protocol for determining bioactivity in rhTNF-α using L929cells was modified to show inhibition of the cytotoxicity of TNF-α. L929cells were grown to near confluency at 37° C. with 5% CO₂ and humidityin a 96-well plate. The cells were plated at 20,000 cells/well for Assay1 and incubated approximately 48 hours. For Assay 2, 40,000 cells/wellwere plated and incubated approximately 24 hours.

Human recombinant TNF-α (Sigma-Aldrich, Saint Louis, Mo.) was preparedat a 4× concentration of 2μg/mL in assay medium (DMEM supplemented with10% FBS; 0.5 μg/mL final concentration in the assay). Next, theanti-TNF-α VHH66 (SEQ ID NO:45) sdAb was serially diluted in assaymedium at 4× concentration. The final assay concentrations using threeseparate batches of anti-TNF-α VHH66 (SEQ ID NO:45) sdAb, designatedBatch A, Batch B, and Batch C. For Assay 1, 10-fold dilutions rangingfrom 100μg/mL to 0.01μg/mL (Batch B and C), and 52 to 0.0052 μg/mL(Batch A). For Assay 2, Batches B and C were tested at a finalconcentration range (2-fold dilutions) of 40μg/mL to 0.3125 μg/mL. Equalvolumes of the TNF-α and the appropriate anti-TNF-α VHH66 (SEQ ID NO:45)sdAb (or assay media) were mixed and incubated for 30 minutes at 37° C.The medium was then aspirated off the cells and replaced with assaymedia containing actinomycin D (final concentration 1μg/mL) or assaymedia only (designated as media only wells). The TNF-α/anti-TNF-α VHH66(SEQ ID NO:45) sdAb mixtures were then added to the appropriate wells inquadruplicate. In addition to the media only control wells (noactinomycin D, no TNF-α), TNF-α (with actinomycin D) and actinomycin Donly (no TNF-α) control wells were included. After incubating for 24hours at 37° C. with 5% CO₂ and humidity, a MTS/PMS solution (Cell Titer96 AQueous Non-Radioactive Cell Proliferation Assay, Promega, MadisonWis.) was added to each well. The plate was incubated for 4 hours at 37°C. with 5% CO₂ and humidity. The absorbance was then read at 490 nm on aVersaMax instrument utilizing SoftMax Pro GxP version 5.4 software fromMolecular Devices (Sunnyvale, Calif.).

Dose response curves were generated and the EC₅₀ value (the value atwhich the effective concentration is half of the maximal) was determinedfor each sdAb. For Assay 1, the EC₅₀ value for Batch A was 2.9μg/mL. TheEC₅₀ value for Batch B was 1.2μg/mL and the EC₅₀ value for Batch C was2.3 μg/mL. The percent cytotoxicity for the control wells and testsamples are provided in Table 61 and 62, respectively.

TABLE 61 Percent Toxicity for Controls (Assay 1) % Toxicity % ToxicitySample Replicate # Sample 1 2 3 4 5 6 7 8 9 10 11 12 Actinomycin D only(no TNFα) 18.6 0 0 0 0 0 0 0 0 5.4 2.3 3.5 Media only (no TNF-α) 0 0 0 00 0 0 0 0 0 0 0 TNFα (0.5 μg/mL) 101.9 102.6 102.7 102.1 101.8 101.8102.1 102.1 101.7 101.6 101.8 102.1

TABLE 62 Percent Toxicity for Test Samples (Assay 1) Standard TestSample Sample Replicate # Mean % Deviation Batch A  52 μg/mL 31.4 28.319.6 23.2 25.6 4.55  5.2 μg/mL 48.4 28.1 30.1 27.3 33.5 8.68 0.52 μg/mL 78.6 70.5 71.4 67.9 72.1 3.97 0.052 μg/mL  101.5 101.1 101.2 101.5 101.30.18 0.0052 μg/mL   102.0 101.6 101.9 102.1 101.9 0.19 Batch B 100 μg/mL15.4 7.7 4.8 5.7 8.4 4.18  10 μg/mL 35.7 34.7 31.6 32.2 33.6 1.70  1μg/mL 34.6 34.4 34.8 33.0 34.2 0.71  0.1 μg/mL 101.2 101.7 101.7 102.0101.7 0.29 0.01 μg/mL  102.1 102.4 102.1 102.3 102.2 0.13 100 μg/mL 15.47.7 4.8 5.7 8.4 4.18 Batch C 100 μg/mL 18.8 15.0 15.7 38.2 21.9 9.50  10μg/mL 34.6 32.8 33.8 32.4 33.4 0.86  1 μg/mL 44.9 42.1 41.6 45.7 43.61.76  0.1 μg/mL 102.0 102.3 102.0 101.7 102.0 0.21 0.01 μg/mL  102.1102.3 102.4 102.1 102.2 0.13

For Assay 2, the concentration range was narrowed in order to furtherrefine the EC₅₀ value. Values of 4.5 μg/mL and 6.2μg/mL were determinedfor Batch B and C, respectively. The percent cytotoxicity for thecontrol wells and test samples are provided in Table 63 and 64,respectively.

TABLE 63 Percent Toxicity for Controls (Assay 2) % Toxicity SampleReplicate # Sample 1 2 3 4 5 6 7 8 Actinomycin D only (no TNFα) 2.3 010.4 --1 0 0 0.4 0 Media only (no TNFα) 0 0 0 0 0 0 0 0 TNFα (0.5 μg/mL)98.0 97.5 97.7 97.4 98 97.4 97.4 97.2 ¹Well was masked as an outlier(Q-test performed).

Percent Toxicity for Test Samples (Assay 1) Standard Test Sample SampleReplicate # Mean % Deviation Batch B 40 μg/mL 12.1 9.6 9.2 11.0 10.51.15 20 μg/mL 10.5 5.7 6.5 7.7 7.6 1.82 10 μg/mL 30.1 31.2 15.1 13.722.5 8.15  5 μg/mL 40.6 39.6 25.1 23.6 32.2 7.90 2.5 μg/mL  96.3 96.284.5 82.6 89.9 6.39 Batch C 40 μg/mL 10.5 6.5 10.6 4.0 7.9 2.79 20 μg/mL5.5 2.6 9.7 2.5 5.1 2.93 10 μg/mL 14.0 11.9 26.6 25.6 19.5 6.63  5 μg/mL44.9 42.5 85.7 86.3 64.9 21.17 2.5 μg/mL  96.9 96.3 97.3 97.3 97.0 0.411.25 μg/mL  97.5 97.3 97.7 97.4 97.5 0.15

Activity against TNF-α was demonstrated for all three batches ofanti-TNFα VHH66 as shown by the reduction in TNF cytotoxicity.Differences between Batch B and C was less than 2-fold. Batch Ademonstrated <2-fold difference as compared to Batch C and slightlyhigher then 2-fold difference compared to Batch B.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. The steps disclosed for the present methods, for example, arenot intended to be limiting nor are they intended to indicate that eachstep is necessarily essential to the method, but instead are exemplarysteps only. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure. All references cited herein are incorporated by reference intheir entirety.

What is claimed is:
 1. A single-domain antibody (sdAb) directed againstan intracellular component, wherein the sdAB can passively cross acellular membrane to target the intracellular component withoutexogenous compounds and without additional membrane targeting sequences.2. The sdAb of claim 1, wherein the intracellular component comprises anucleic acid, protein, lipid, carbohydrate, or combination thereof. 3.The sdAb of claim 1, wherein the intracellular component comprises aprotein.
 4. The sdAb of claim 3, wherein the protein isun-phosphorylated.
 5. The sdAb of claim 3, wherein the protein isphosphorylated.
 6. The sdAb of claim 1, wherein the intracellularcomponent comprises a STAT protein.
 7. The sdAb of claim 6, wherein theSTAT protein comprises STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, orSTAT6.
 8. The sdAb of claim 1, wherein the sdAb is multispecific for twoor more antigens.
 9. A method of treating a disease, preventing adisease or preventing the reoccurrence of a disease in a subject usingthe sdAb of claim
 1. 10. The method of claim 9, wherein the sdAb can beused to deliver a therapeutic agent.
 11. The method of claim 10, whereinthe sdAb can be used to deliver the therapeutic agent across theblood-brain barrier to an individual in need thereof.
 12. The method ofclaim 9, wherein the sdAb is used in combination with one or morecompounds.
 13. The method of claim 12, wherein the one or more compoundsis a transcriptional inhibitor.
 14. A single-domain antibody (sdAb)directed against an intracellular component, wherein the sdAB canpassively cross blood-brain barrier to target the intracellularcomponent without exogenous compounds and without additional membranetargeting sequences.
 15. The sdAb of claim 14, wherein the intracellularcomponent comprises a nucleic acid, protein, lipid, carbohydrate, orcombination thereof.
 16. The sdAb of claim 14, wherein the intracellularcomponent comprises a protein.
 17. The sdAb of claim 16, wherein theprotein is un-phosphorylated.
 18. The sdAb of claim 16, wherein theprotein is phosphorylated.
 19. The sdAb of claim 14, wherein theintracellular component comprises a STAT protein.
 20. The sdAb of claim19, wherein the STAT protein comprises STAT1, STAT2, STAT3, STAT4,STAT5a, STAT5b, or STAT6.
 21. The sdAb of claim 14, wherein the sdAb ismultispecific for two or more antigens.
 22. A method of treating adisease, preventing a disease or preventing the reoccurrence of adisease in a subject using the sdAb of claim
 14. 23. The method of claim22, wherein the sdAb can be used to deliver a therapeutic agent to anindividual in need thereof.
 24. The method of claim 22, wherein the sdAbis used in combination with one or more compounds.
 25. The method ofclaim 22, wherein the one or more compounds is a transcriptionalinhibitor.